Doctoral Degrees (Food Science)

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Browsing Doctoral Degrees (Food Science) by browse.metadata.advisor "Gouws, Pieter Andries"

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    Antibiotic resistant bacteria prevalent in livestock and wildlife species in South Africa
    (Stellenbosch : Stellenbosch University, 2019-04) Van den Honert, Michaela Sannettha; Gouws, Pieter Andries; Hoffman, Louwrens C.; Stellenbosch University. Faculty of Agrisciences. Dept. of Food Science.
    ENGLISH ABSTRACT: Much research has focussed on the fate of antibiotics in clinical settings whereas research of antibiotics in natural environments has been comparatively limited. It has been hypothesised that wildlife could play a significant role in the development of antibiotic resistant bacteria in nature as a variety of wildlife species carry antibiotic resistant bacteria and cover a large territory throughout their lifespan The aim of this study was to determine whether wild ungulates, namely, African buffalo (Syncerus caffer), black wildebeest (Connochaetes gnou), blue wildebeest (Connochaetes taurinus), bontebok (Damaliscus pygargus), eland (Taurotragus oryx), fallow deer (Dama dama), impala (Aepyceros melampus) and springbok (Antidorcas marsupialis), host antibiotic resistant bacteria, specifically, Escherichia coli, Enterococcus faecalis and Staphylococcus aureus, from various South African farms. The Kirby-Bauer disk diffusion method was used according to the Clinical and Laboratory Standards Institute 2018 guidelines. Overall, antibiotic resistance among the wild ungulate species was low towards the selected antibiotics. On average, the antibiotic resistance levels were 8% E. coli (N= 353), 4% E. faecalis (N= 194) and 22% S. aureus (N= 106). The highest antibiotic resistance was towards antibiotics which are of natural origin, namely the β-lactams and streptomycin. These antibiotics are found in the soil microbiome, produced by Actinobacteria. In addition, certain resistant genes were detected using the polymerase chain reaction in isolates which showed phenotypic resistance. The resistant genes sul1 (40%), sul2 (80%), sul3 (0%), blaCMY (98%), tetA (63%), tetB (75%), tetC (0%) and aadA (98%) were detected in resistant E. coli isolates (N= 44); tetK (7%), tetL (100%), tetM (100%), blaZ (100%), vanA (95%) and vanB (10%) in resistant S. aureus (N= 5) and E. faecalis (N= 22) isolates. The results of this study indicate that wildlife can be considered a natural reservoir of antibiotic resistant genes. The wildlife were also found to be more multi-drug resistant than the livestock. Thus it is speculated that these resistant genes are picked up from the soil and the surrounding environment and are spread by the animals as well as by other natural vectors like the wind and flies. Various factors and agricultural practices were found to influence the antibiotic resistance of the bacteria harboured by the wildlife species, namely, co-grazing with livestock, the practice of wildlife supplementary feeding and farm history of antibiotic use. Bacteria isolated from game meat was frequently more antibiotic resistant than bacteria from the faeces, indicating human cross-contamination during slaughter. The level of antibiotic resistance determined in this study from the bacteria of the wildlife from pristine areas, could serve as a baseline for monitoring the influence of human activities on the development of antibiotic resistance in various environments, which this study contributed towards.
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    The efficacy of bacteriophages FO1a and S16 in the reduction of Salmonella on chicken carcasses in a South African poultry processing environment
    (Stellenbosch : Stellenbosch University, 2022-12) Wessels, Kirsten; Gouws, Pieter Andries; Rip, Diane; Stellenbosch University. Faculty of AgriSciences. Dept. of Food Science.
    ENGLISH ABSTRACT: Much of the research surrounding bacteriophages (phages) as a processing aid for the control of Salmonella on chicken meat has been conducted in vitro in the laboratory. Information about the efficacy and application of bacteriophages as part of a hurdle concept in the chicken processing environment is limited. In South Africa, the use of certain antibiotics in live broilers and the use of chlorine-containing antimicrobials in the processing environment, are still permitted as Salmonella control methods. The incidence of Salmonella in chicken meat in South Africa is unclear, but previous research has repeatedly shown that the use of antibiotics and/or chlorine selects for resistance in Salmonella. The aim of this study was to determine the efficacy of a commercial phage cocktail PhageGuard S™ (PGS) (FO1a and S16 phages) in the reduction of Salmonella on chicken carcasses through a validated spraying system in a South African chicken processing plant. This study also investigated the incidence and antibiotic susceptibility profiles of Salmonella isolated from chicken carcasses in the plant. The PGS was applied at a 1% (v/v) concentration onto chicken carcasses via a spraying system (validated specifications: 530 µm nozzle diameter, 200 mesh strainer and 3 Bar pump pressure) after the chlorine spin chilling step. Neck skins samples were collected before the inside- outside wash step (N= 80) and after the PGS application step (N= 160). The neck skin samples were tested for Salmonella presence/absence (EN ISO 6579/A1 (02/2006)) and confirmed using Vitek®. Confirmed Salmonella isolates were screened for antibiotic susceptibility using the Kirby-Bauer disk diffusion method according to M100 from the Clinical and Laboratory Standards Institute (CLSI, 2020). Confirmed Salmonella isolates from neck skin samples collected after PGS application were re-exposed to PGS in the laboratory via a killing assay (Micreos Food Fafety, NL) to determine if the isolates were resistant to the PGS. Before the inside-outside step the Salmonella incidence was 60% with a large portion of these isolates showing resistance to tetracycline (56.3%) and sulfonamide (43.8%). After the combination of the inside-outside wash step, chlorine spin chilling and PGS application, the Salmonella incidence decreased to 23.75%, where more than half of these isolates showed resistance to tetracycline (63.2%) and sulfonamide (55.3%). For the killing assay, all isolates which survived PGS in the processing environment were reduced by 100% in the laboratory, highlighting that the phages were unable to reach the Salmonella via the spray application, and not that the Salmonella was resistant to the phages. The results in this study showed that the multi-drug resistant Salmonella in the chicken neck skins survived a complete immersion in chlorine but were successfully reduced by PGS, making phages a potential solution to many persistent microbial problems. This study also provides valuable insight into implementing phages into the large-scale hurdle concept of a processing environment and highlights the importance of the application method to ensure safe delivery of the phages to the target bacteria for a high efficacy.
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    The prevalence of Campylobacter and Arcobacter species in ostriches from South Africa
    (Stellenbosch : Stellenbosch University, 2020-04) Shange, Nompumelelo; Gouws, Pieter Andries; Hoffman, Louwrens C.; Stellenbosch University. Faculty of AgriSciences. Dept. of Food Science.
    ENGLISH ABSTRACT: The overall aim of this thesis was to determine the prevalence of Campylobacter and Arcobacter species in ostriches from South Africa. In humans Campylobacter and Arcobacter species can cause of gastroenteritis, Guillian Barré syndrome, septicaemia and bacteraemia. Previous research has indicated that the consumption of contaminated poultry meat is the main route of infection for humans and by extension poultry species are deemed primary reservoirs of Campylobacter and Arcobacter species. Currently, there is a lack of information regarding Campylobacter and Arcobacter species in relation to ostriches from South Africa. Artificially and naturally reared ostrich chicks at the age of 2, 4, 6 and 12 weeks were sacrificed, and caeca samples were excised. Campylobacter spp. (C. jejuni) was detected in artificially reared chicks, on the 12th week. A persistent presence of Arcobacter (A. skirrowii) was detected from the 2nd until 12th week of life for both artificially and naturally reared ostrich chicks. Additionally, cohorts that belonged to the same batch as the sacrificed ostrich chicks, regardless of the rearing process were sampled at the slaughter age of 10 and 12 months. Arcobacter spp. (A. skirrowii) and Campylobacter spp. (C. jejuni) were isolated from 56-70% of slaughter age birds. Cloacal swabs were also obtained from live ostriches reared on 30 different farms situated in South Africa (Oudtshoorn). Cloacal swabs were processed with family specific PCR (n = 168 pooled cloacal swabs), the Cape Town protocol (n = 836 cloacal swabs), ISO 10272-1:2006 (n = 836 cloacal swabs) and a selective Arcobacter spp. method (n = 415 cloacal swabs). Family specific PCR determined an average prevalence of 24.63%. The ISO 10272-1:2006 method and Cape Town Protocol determined a prevalence of 16.83% and 0% for Campylobacterspp., respectively. For Arcobacter spp. a prevalence of 18.80% and 39.14% was determined with the Cape Town protocol and selective Arcobacter spp. method, respectively. Higher prevalence levels were determined when ostriches were sampled during spring and autumn, respectively. Higher prevalence levels were also detected in ostriches reared on farms that made use of borehole water. Higher prevalence levels were seen for ostriches reared on farms with wild water birds. During slaughter, Arcobacter spp. were detected at a prevalence level of 73% at post-skinning. At post-evisceration, 73% and 83% of samples were contaminated with Campylobacter spp. and Arcobacter spp., respectively. At post-chilling, 66% and 67% were contaminated with Campylobacter spp. and Arcobacter spp., respectively. Additionally, a second study to evaluate the occurrence of Campylobacter spp. and Arcobacter spp. was conducted to see whether routine testing was required for abattoirs. E. coli and coliforms were also enumerated to determine the occurrence of faecal contamination during slaughter. Overall, a low occurrence of Campylobacter spp. (0.98% and 0%), Arcobacter spp. (1.31% and 1.64%), E. coli (0.13 log cfu/g) and coliforms (0.53 log cfu/g) was determined for all three abattoirs. Antibiotic resistance in Campylobacter spp. and Arcobacter spp. isolated from ostriches and ostrich meat was determined. Campylobacter spp. and Arcobacter spp. isolates were generally resistant to antibiotics in the following order cephalothin, vancomycin and erythromycin and tetracycline. The majority of Campylobacter spp. (92.86%) and Arcobacter spp. (80.95%) isolates exhibited multi-drug resistance. Overall, this research shows that ostriches from South Africa can be considered as potential carriers of species belonging to the Campylobacteraceae family and infection can occur at young age. Carcasses can be contaminated during slaughter and species carried by ostriches can be resistant to essential antibiotics; ultimately highlighting the need for routine testing of Campylobacter and Arcobacter species.
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    Profiling of traditional South African biltong in terms of processing, physicochemical properties and microbial stability during storage
    (Stellenbosch : Stellenbosch University, 2017-03) Jones, Maxine Sylvia; Hoffman, Louwrens C.; Gouws, Pieter Andries; Arnaud, E.; Stellenbosch University. Faculty of AgriScience. Dept. of Food Science.
    ENGLISH SUMMARY: In South Africa, there are no processing guidelines for biltong production and therefore the industry uses different processing parameters which results in variation in the product. The same process was used throughout this study and drying was done using constant parameters – temperature 25 ± 2°C, relative humidity 30 ± 5%, air velocity 2 ± 0.2 m/s. An initial study investigating the influence of vinegar addition during the production of beef biltong showed that vinegar addition does not influence its drying kinetics. The biltong reached a 50% weight loss after 66 hours and a 65% weight loss after 96 hours. The use of different meat muscles (topside, semimembranosus and silverside, biceps femoris), beef with subcutaneous fat and gemsbok (Oryx gazelle) showed differences (p ≤ 0.05) in drying rates when dried to a targeted weight loss of 65%. The two lean beef muscles both dried in 96 hours. The gemsbok topside took only 78 hours with a drying pattern similar to the lean beef topside. The fatty beef topside took 118 hours to dry. The microbiological profile of beef biltong over a three month shelf-life storage were studied. Final weight loss during drying and packaging method (modified atmospheric packaging and vacuum packaging) did not have an effect (p > 0.05) on the microbiological profile. Total viable counts and coliforms were only reduced in biltong with vinegar added. After drying, yeasts and moulds were already present at high levels (~ 2.5 log cfu.g-1) but not visible. After six weeks, yeasts and moulds became visible. Staphylococcus aureus was present at less than 20 log cfu.g-1 while Listeria monocytogenes, Salmonella spp. and Escherichia coli were not present during the three month storage period. Yeast and mould growth on biltong is a problem and therefore a challenge study was included. Beef biltong produced without and with vinegar and dried to a 50% or 65% weight loss were inoculated with yeasts and moulds. No yeast and mould growth was seen on biltong with vinegar but 1.8 – 2.5 log cfu.g-1 was detected after 34 days. Biltong without vinegar showed yeast and mould growth after 10 days with levels of 2.8 – 3.1 log cfu.g-1. Saccharomyces spp. (yeast) and Aspergillus spp., Fusarium spp. and Penicillium spp. (moulds) were the most common yeast and moulds. A small-scale study using ultrasound in the salting step of beef biltong processing showed that ultrasonic-brining did not have an effect on either the salting or drying kinetics contrary to what was expected. Throughout the study the physicochemical properties of the beef biltong gave consistent results. An approximate 50% and 65% weight loss produced biltong with a moisture content of 50% and 30%, respectively and water activity of 0.74 – 0.78 and 0.81 and 0.86, respectively. Weight loss or the addition of vinegar did not play a role in the salt content (dry basis). Beef biltong without vinegar had a pH 5.56 – 5.75 while the addition of vinegar to biltong lowered the pH of biltong to 4.89 – 4.93. It is recommended that the biltong industry should standardise their drying parameters to avoid variation in quality and for a more microbial stable product. Vinegar could be added as it has an effect on the yeast and mould growth. Biltong with water activity ranging from 0.74 to 0.83 does not have a shelf-life of more than three months when using modified atmosphere packaging or vacuum packaging. The data generated in this study serves as a base-line for future studies focused on optimising and standardising the drying procedures applicable to biltong.
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    Taxonomy of species of Alicyclobacillus from South African orchards and fruit concentrate manufacturing environments and the prevention of fruit juice contamination
    (Stellenbosch: University of Stellenbosch, 2009-12) Groenewald, Willem Hermanus; Witthuhn, R. C.; Gouws, Pieter Andries; University of Stellenbosch. Faculty of Agrisciences. Dept. of Food Science.
    ENGLISH ABSTRACT: Species of Alicyclobacillus are acid-tolerant and heat-resistant bacteria that cause spoilage of heat-treated fruit juices stored at room temperature. During the past decade, Alicyclobacillus spp. have become a major cause of spoilage in pasteurised fruit juices leading to significant economic losses world-wide. Spoilage has been reported in apple, pear, orange, peach, mango and white grape juice, as well as in fruit juice blends, fruit juice containing drinks and tomato products, such as tomato juice and canned tomatoes. Spoilage is characterised by a medicinal smell and guaiacol production. These endospore-formers have been shown to survive pasteurisation conditions of 95 °C for 2 min, grow at temperatures between 25° and 60 °C and a pH range of 2.5 to 6.0. Knowledge of this organism is limited, both locally and internationally and the route of contamination to the final product is not well established. In this study the fruit concentrate processing environment was investigated as a potential source and route of contamination for the final product. Species of Alicyclobacillus were isolated from orchard soil, various stages during processing and from fruit juice and concentrates. The isolates were identified based on morpholological, biochemical and physiological properties. Identification to species level was done by 16S ribosomal RNA gene sequencing and strain differentiation by RAPD-PCR. Results indicate that species of A. acidoterrestris and Alicyclobacillus acidocaldarius were found in orchard soil and throughout the processing environment. This is the first report on the isolation of these species from orchard soil, vinegar flies and the fruit processing environment. The 16 isolates identified as A. acidoterrestris grouped into four clusters based on RAPD-PCR banding patterns, suggesting that they belong to at least four genotypic groups. Isolates from the fruit concentrate, wash water and soil located outside of the fruit processing plant grouped into one cluster. Concluded from these results, A. acidoterrestris found in the wash water and soil outside of the factory could act as a potential reservoir of organisms for the contamination of the final fruit concentrate. Thus good manufacturing practices play an essential role in controlling incidence of spoilage caused by these bacteria. Fruit juices can be treated using ultraviolet (UV-C) light with a wavelength of 254 nm, which has a germicidal effect against micro-organisms. Alicyclobacillus acidoterrestris spores were inoculated into tap water, used wash water from a fruit processing plant and grape juice concentrate. Ultraviolet dosage levels (J L−1) of 0, 61, 122, 183, 244, 305 and 367 were applied using a novel UV-C turbulent flow system. The UV treatment method was shown to reliably achieve in excess of a 4 log10 reduction (99.99%) per 0.5 kJ L-1 of UV-C dosage in all the liquids inoculated with A. acidoterrestris. The applied novel UV technology could serve as an alternative to thermal treatments of fruit juices for the inactivation of Alicyclobacillus spores or in the treatment of contaminated processing wash water. Finally, the thermal inactivation at 95 °C for two strains of A. acidoterrestris isolated from contaminated fruit juice concentrates were investigated in a 0.1% (m/v) peptone buffer solution (pH 7.04) and grape juice (pH 4.02, 15.5 °Brix). The thermal inactivation of A. acidoterrestris spores followed first-order kinetics, suggesting that as the microbial population is exposed to a specific high temperature, the spores inactivated at a constant rate. D-values determined in the buffer solution were calculated to be 1.92 min and 2.29 min, while in grape juice D-values were found to be 2.25 min and 2.58 min for the two strains tested. From this study it is clear that the D-value is dependant on the strain tested, but also on the soluble solids of the solution the cells are suspended in. The results indicated that the spores of A. acidoterrestris isolated from South African fruit juice concentrate may survive after the pasteurisation treatment commonly applied during manufacturing.