The aim of the present work was to investigate aflatoxin levels in various food commodities and to study its production by Aspergillus parasiticus in culture to find out the possible ways to control it. Of 40 food samples collected from Abha region, Saudi Arabia, only 25% were contaminated with aflatoxins. Oil-rich commodities had the highly contaminated commodities by fungi and aflatoxins while spices were free from afatoxins. The optimal pH for the growth of A. parasiticus and its productivity of aflatoxin B, was found at 6.0, while the best incubation conditions were found at 30°C for 10 days. D-glucose was the best carbon source for fungal growth, as well as aflatoxin production. Corn steep liquor, yeast extract and peptone were the best nitrogen sources for both fungal growth and toxin production. (NH4)2HPO4 (1.55 gL-1) and NaNO2 (1.6 gL-1) reduced fungal growth and toxin production with 37.7% and 85%, respectively. Of ten amino acids tested, asparagine was the best for aflatoxin B, production. Zn2+ and Co2+ supported significantly both fungal growth, as well as aflatoxin B, production at the different tested concentrations. Zn2+ was effective when added to A. parasiticus growth medium at the first two days of the culture age. The other tested metal ions gave variable effects depending on the type of ion and its concentration. Water activity (a ) was an important factor controlling the growth of A. parasiticus and toxin production. The minimum aW for the fungal growth was 0.8 on both coffee beans and rice grains, while aW, of 0.70 caused complete inhibition for the growth and aflatoxin B, production. H202 is a potent inhibitor for growth of A. parasiticus and its productivity of toxins. Incubation with NaHCO3 and C6H5000Na converted aflatoxin B, to a water-soluble form which returned to aflatoxin B, by acid treatment. Black pepper, ciliated heath, cuminum and curcuma were the most inhibitory spices on toxin production. Glutathione, quinine, EDTA, sodium azide, indole acetic acid, 2,4-dichlorophenoxy acetic acid, phenol and catechol were inhibitory for both growth, as well as, aflatoxin B, production. Stearic acid supported the fungal growth and decreased the productivity of AFBI gradually. Lauric acid is the most suppressive fatty acid for both fungal growth and aflatoxin production, but oleic acid was the most potent supporter. Vitamin A supported the growth but inhibited aflatoxin B, production. Vitamins C and D2 were also repressive particularly for aflatoxin production. The present study included determining the activities of some enzymes in relation to aflatoxin production in A. parasiticus culture during 20 days. Glycolytic enzymes and pyruvate-generating enzymes seems to be linked with aflatoxin B, production. Also, pentose-phosphate pathway enzymes may provide NADPH for aflatoxin B, synthesis. The decreased activities of TCA cycle enzymes particularly from 4th day of growth up to 10th day were correlated with the increase of aflatoxin B, production. All the tested enzymes as well as aflatoxin B, production were inhibited by either catechol or phenol.

No description available.

M.Sc. (Nanoscience) / Ochratoxin A and Aflatoxin B1 are important food contaminates as they are known to be mutagenic, genotoxic, nephrotoxic, hepatotoxic, immunosuppressive and teratogenic to both animals and humans. These mycotoxins are associated with the contamination of food stuff such as grapes, maize, red pepper, meat, milk, beans and processed products from contaminated raw material. Current physical, biological and chemical methods employed to improve the safety of food often compromise the nutritional value and result in huge losses. The alternative to these treatments are addition of supplements with protective properties to reduce the toxicity of mycotoxins or prevent their formation. The work presented in this dissertation reports an attempt to develop such materials to prevent damage caused by ochratoxin A and aflatoxin B1. This was done through the synthesis; characterisation and cytotoxicity study of chitosan nanoparticles with methanolic plant extracts (L. leonurus, M. longifolia and A. montanus). Inhibition of cellular damage due to mycotoxins for possible application in prevention of cellular damage by mycotoxins also presented. Chitosan nanoparticles were synthesised using an ionic gelation method with sodium triphosphate as the cross linker. The methanolic medicinal plants extracts were incorporated into the chitosan solution before synthesising nanoparticles, and nanoparticle synthesis initiated by the addition of sodium triphosphate solution. The synthesised products were characterised using zetasizer, transmission electron microscopy, x-ray diffraction and Fourier-transform infrared spectroscopy. The extracts’ antioxidant ability was evaluated before incorporation into chitosan using 2, 2-diphenyl- 1-picrylhydrazy (DPPH) radical scavenging assay. This assay was performed using UVvis spectroscopy. The cytotoxicity of the synthesised nanoparticles was assessed using a Vero cell line and by evaluating the cell viability with an MTS assay. The nanoparticles were successfully synthesised and showed the presence of different functional groups as expected. Plain chitosan nanoparticles were roughly spherical shaped and had smooth surfaces, nanoparticles containing extracts similarly were spherical in shape as well but had rougher surfaces when visualised under TEM. All nanoparticles had positive zeta potentials between 26 – 28 mV. The average particle sizes ranged between 31 – 65 nm as measured using TEM and average particle sizes obtained using zetasiser was 78 – 190 nm. The cytotoxicity studies of plain nanoparticles and nanoparticles with extract showed that the synthesised nanomaterials were not toxic even at concentration of 500 μg/ml and less than 20% of the Vero cells were affected under these conditions.

Quality control procedures during food processing may involve either lengthy enrichment steps, precluding enumeration of bacteria in contaminated food, or direct inoculation of food samples onto appropriate selective media for subsequent enumeration. However, sublethally injured bacteria often fail to grow on selective media, enabling them to evade detection and intervention measures and ultimately threaten the health of consumers. This study compares traditional selective and nonselective agar-based overlays versus two commercial systems (Petrifilm and Easygel) for recovery of injured Escherichia coli B-41560, originally an isolate from ground beef. Bacteria were propagated in tryptic soy broth (TSB), ground beef, or infant milk formula (IMF) to a density of 106-108 CFU/mL, and stressed for six minutes either in lactic acid (pH of 4.5) or heat-shocked for 3 min. at 60°C. Samples were pour- plated in basal layers of either tryptic soy agar (TSA), Sorbitol MacConkey (SMAC), or Violet Red Bile (VRB) agar and resuscitated for 4h prior to addition of agar overlays. Other stressed bacteria were plated directly onto the commercial media Petrifilm and Easygel. Our results indicate that the use of selective and nonselective agar overlays for sensitive recovery and accurate enumeration of E. coli B-41560 is dependent on the stress treatment and food system. These data underscore the need to implement food safety measures that address sublethally- injured bacteria such as E. coli O157:H7, without the use of enrichment steps, in order to avoid underestimation of true densities for target pathogens. / Department of Biology

Thesis (MSc Food Science)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: South Africa is a water scarce country and farmers are forced to irrigate crops with river water. Contamination of South African rivers has been reported and the carry-over of bacteria from river water to produce has been confirmed. Foodborne outbreaks linked to fresh produce are increasing world-wide. A total of 151 fresh produce samples (lettuce, tomatoes, beans, peas, coriander, basil, mint, rocket, thyme, spinach, cabbage, parsley and sprouts) were sourced from small-scale and commercial farms, farmers’ markets and retail outlets. Total coliforms (TC) and E. coli loads on the produce were determined with Colilert-18. Isolates were phenotypically characterised and identified with the API system and the E. coli identification confirmed with uidA PCR. Sixty-three E. coli isolates were identified. Three were not identified as E. coli with the API system but were positive for the uidA gene. The TC loads for the produce from the farms, farmers’ markets and retail outlets were all in the range of log 3 to log 8.38 MPN.100 mL-1. Escherichia coli was found to be most prevalent on produce samples from farmers’ markets with the highest E. coli load (log 7.38 MPN.100 mL-1) on cabbage sampled from a commercial farm. Escherichia coli were present on 8% of the produce samples. The maximum TC and E. coli loads found on the fresh produce were log 8.38 and log 7.38 MPN.100 mL-1, respectively. The lowest risk in terms of TC and E. coli presence and load was observed on fresh produce from retail outlets and the highest risk was on fresh produce from farmers’ markets. Phenotypic dendrograms and a PCA plot were statistically constructed to determine similarity groupings of the isolates and three main E. coli clusters were formed. These three clusters could not be directly linked to a specific produce type or source type. A larger variation E. coli phenotypes was observed present on fresh produce within the three clusters. All E. coli isolates were also subjected to triplex and multiplex PCR analysis to identify their phylogenetic groups and the presence of INPEC and ExPEC strains. Fourteen isolates belonged to genotypic group A0, 11 to A1, 20 to B1, 7 to B23 and 11 to D2. Thus a large variation E. coli genotypes are present but it cannot be linked to a specific source type or produce type. Multiplex PCR testing for INPEC revealed that none of the E. coli isolates were carriers of the INPEC genes. The isolates were also tested for the presence of ExPEC gene sequences: papA, papC, sfa/foc, iutA, kpsMT II and afa/dra. None of the isolates were classified as ExPEC (which required the presence of two or more genes) but three of the isolates did test positive for the presence of the kpsMT II gene. The latter could indicate that potentially pathogenic E. coli can be evolving in the environment and increase the risk of pathogenic E. coli occurring on fresh produce. In conclusion, the presence of E. coli (commensal or pathogenic) on fresh produce is unacceptable according the South African Department of Health. According to this study the identification of E. coli types could not be correlated with the presence of E. coli on the different produce types and thus the presence of E. coli on fresh produce is unpredictable. It is recommended that extensive safety precautions should be in place throughout every step in the production chain from harvest to the consumer’s kitchen to reduce the probability of contamination of fresh produce. / AFRIKAANSE OPSOMMING: Suid-Afrika is ‘n waterskaars land en boere word gedwing om rivier water te gebruik vir gewas besproeiing. Kontaminasie van Suid-Afrikaanse riviere is al telkemale aangemeld en die oordrag van bakterieë vanaf rivierwater na vars produkte is al voorheen bevestig. Voedselverwante uitbrake wat gekoppel is aan vars produkte is besig om wêreldwyd toe te neem. ‘n Totaal van 151 vars produk monsters (blaarslaai, tamaties, boontjies, ertjies, koljander, basilie, kruisement, roket, tiemie, spinasie, kool, pietersielie en spruite) was verkry van klein-skaalse en kommersiële plase, plaasmarkte en kettingwinkels. Totale kolivorme (TK) en E. coli tellings op die vars produkte is bepaal deur middel van Colilert-18. Isolate word fenotipies gekarakteriseer en geïdentifiseer met die API sisteem en die E. coli identifikasie is bevestig met uidA PKR. Drie-en-sestig E. coli isolate is geïdentifiseer. Drie is nie met met die API sisteem as E. coli geklassifiseer nie, maar was wel positief vir die uidA geen. Die TK tellings vir die vars produkte van die plase, plaasmarkte en kettingwinkels was almal in die reeks van log 3 tot log 8.38 MPN.100 mL-1. Escherichia coli teenwoordigheid was die meeste op groente monsters van plaasmarkte, maar die hoogste E. coli telling (log 7.83 MPN.100 mL-1) was op ‘n kool monster van ‘n kommersiële plaas. Escherichia coli was teenwoordig op 8% van die vars produk monsters. Die maksimum TK en E. coli wat teenwoordig was op die vars produkte was log 8.38 en log 7.38 MPN.100 mL-1 onderskeidelik. Die laagste risiko in terme van TK en E. coli teenwoordigheid en tellings is waargeneem op vars produkte van kettingwinkels en die hoogste risiko is op vars produkte van plaasmarkte. Fenotipiese dendrogramme en ‘n PKA plot is statisties gekonstrueer om ooreenstemende groepe van isolate te identifiseer en drie hoof groepe is gevorm. Daar kon geen direkte verband gevind word tussen hierdie drie groepe en ‘n spesifieke produk-tipe of ‘n spesifieke bron-tipe nie. ‘n Groter variasie in E. coli fenotipes teenwoordig op die vars produkte is waargeneem binne die drie groepe. Alle E. coli isolate was onderworpe aan tripleks en multipleks PKR analise om die filogenetiese groep van elke isolaat te bepaal en of enige INPEC of ExPEC stamme teenwoordig is. Veertien isolate behoort aan genotipiese groep A0, 11 aan A1, 20 aan B1, 7 aan B23 en 11 aan D2. Dus is ‘n groot variasie E. coli genotipes teenwoordig maar dit kan nie gekoppel word aan ‘n spesifieke produk-tipe of bron-tipe nie. Multipleks PKR analise vir INPEC het gewys dat geeneen van die E. coli isolate enige INPEC gene dra nie. Die isolate is ook getoets vir die teenwoordigheid van ExPEC geen volgordes: papA, papC, sfa/foc, iutA, kpsMT II en afa/dra. Geeneen van die isolate is geklassifiseer as ExPEC (wat die teenwoordigheid van twee of meer gene vereis) nie, maar drie van die isolate het wel positief getoets vir die teenwoordigheid van die kpsMT II geen. Laasgenoemde kan ‘n aanduiding wees dat potensiële patogeniese E. coli in die omgewing kan ontwikkel en dus dan die risiko van die teenwoordigheid van patogeniese E. coli op vars produkte sal verhoog. Ter afsluiting, die teenwoordigheid van E. coli (nie-patogenies en patogenies) op vars produkte is onaanvaarbaar volgens die Suid-Afrikaanse Departement van Gesondheid. Volgens hierdie studie kan die identifisering van E. coli tipes nie gekorreleer word met die teenwoordigheid van E. coli op verskillende produk-tipes nie en dus is die teenwoordigheid van E. coli op vars produkte onvoorspelbaar. Dit word aanbeveel dat ekstensiewe voorsorgmaatreëls in plek moet wees in elke stap dwarsdeur die produksie ketting, vanaf oestyd tot in die verbruiker se kombuis, om die moontlikheid van vars produk kontaminasie te verminder.

[Truncated abstract] The applications of biosensors are rapidly expanding with the increased emphasis placed on the use of technology in the evaluation of food safety and also in military use. The United States food industry carried out 144.3 million microbiological tests in 1999 (Alocilja and Radke, 2003). These numbers are expected to rise with the recently implemented regulatory measures for food safety in the United States. In fact, similar trends in food safety are occurring on a global scale. Furthermore, with the recognition and establishment of Microbial Forensics as a new field of forensics, the interest in biosensor development for the detection of microbes will thrive. Moreover, the recent spate of biocrimes, notably the anthrax scares, has called for newer and improved techniques for the sensitive, rapid and reproducible detection of microbes. Biosensors have the capability to fill this role as an efficient device for microbial detection. There is a wide range of biosensors available for different purposes. In addition, their versatility allows for their overlap in many fields. The quartz crystal microbalance (QCM) is a biosensor that is cost-efficient, sensitive, field-deployable with the ability to perform automated, real-time assays within minutes. The QCM is a mass sensitive device that works on the principle where a change in mass deposited on the crystal is inversely proportional to the change in the resonant frequency of the crystal. Therefore, frequency decreases with increasing mass deposited. The QCM has been used in several studies as a biosensor for the detection of a number of viral and bacterial species. ... High antibody incubation concentration required a shorter antibody incubation duration. Conversely, low antibody incubation concentration required a longer antibody incubation duration. Furthermore, regardless of antibody incubation concentration, a distinct pattern in the rate of antibody binding with time was observed. One hour antigen incubation at ambient room temperature (22.5oC) was sufficient for the efficient binding of the antigens to the immobilized antibody layer. Extension of antigen binding time to 15 hours produced inconsequential differences in readings. The binding efficiency of the quartz crystals after a storage period of 2 to 4 weeks at ambient room temperature (22.5oC) fared better than the crystals that were refrigerated at 4oC. Results showed that 0.2M glycine hydrochloride is a poor reagent for the removal of the antigen layer on the quartz crystals for repeated assay use. The 16-mercaptohexadecanoic acid (MHDA) layer and adsorbed proteins on the quartz crystals can be removed by a mixture of sulphuric acid and hydrogen peroxide, known as a piranha process. This allows the crystals to be repeatedly recoated and reused. Overall, this research provides new insights into the preparation process of the quartz crystals for the specific detection of E. coli O157:H7. Conclusive results have been obtained for several tested parameters and suggestions have been raised for further studies in the optimization of the QCM for the E. coli O157:H7 detection process. With improved knowledge and recognition in the capability of the QCM as a biosensor, the QCM may soon be used in conjunction with conventional techniques for the rapid detection of E. coli O157:H7.

Thesis (M. Tech.) -- Central University of Technology, Free State, 2010 / South Africa currently faces one of the highest HIV prevalence rates in the world. As this prevalence rises, the strain placed on its hospitals is likely to increase due to the shortage of beds. The devastating effects of HIV/AIDS initiated the establishment of a hospice which is a non-governmental organisation whose goal is the provision of care for terminally ill patients, either in their homes, in hospitals or in a hospice’s own in-patients wards. Part of the hospice’s mission is to offer palliative care without charge to anyone who requires it. The basic elements of hospice care include pain and symptom management, provision of support to the bereaving family and promoting a peaceful and dignified death. This also includes the provision of cooked foods to the patients using the kitchen facilities of the hospices for this activity. It is well known that the kitchen is particularly important in the spread of infectious disease in the domestic environment due to many activities that occur in this particular setting. Food and water safety is especially important to the persons infected with the human immunodeficiency virus (HIV) or with immunodeficiency syndrome (AIDS).It is estimated that food-borne pathogens (disease–causing agents) are responsible for 76 million illnesses, some resulting in death, in the United States alone every year. In one study of patients with AIDS, two-thirds had diarrhoeal disease and in two-thirds of these, the following enteric pathogens were identified: Salmonella, Shigella, Listeria, Yersnia, Cryptosporidium, Entamoeba histolylica and Campylobacter sp. In an epidemiological study of patients with HIV infection a close association was found between consumption of raw or partially cooked fish and antimicrobial-resistant Mycobacterium avium complex. Antibiotic resistance in food-borne pathogens has become a reality and this poses a serious threat to the medical fraternity since it diminishes the effectiveness of treatment. This study was undertaken to determine the prevalence of foodborne pathogens including bio aerosols isolated from the kitchen surfaces and food handler’s before and after cooking. The antibiotic resistance of the isolated pathogens was further determined to assess their impact on treatment. The following microbiota were isolated: Total viable counts (TVC), Coliforms, Escherichia coli, Staphylococcus aureus, Pseudomonas and presumptive Salmonella. The hospices had high counts of E.coli and S.aureus on the cutting boards for the breakfast session compared to the traditional home based kitchens. It was speculated that this could have originated from crosscontamination via the foodhandler’s hands and the food served. It is evident from the results that hospices lack a management system regarding the prevalence of E. coli as it was present on the cutting boards throughout the food preparation sessions. Gram negative organisms (coliform and P. aeruginosa) were in particular both resistant to oxacillin and this pose a great challenge in this particular setting. This can be addressed by putting emphasis on hygiene as a strategy per se for reducing antibiotic resistance.