Thesis (MSc)--University of Stellenbosch, 2007. / ENGLISH ABSTRACT: The World Health Organisation (WHO) estimates that at least 75% of infants receive infant formula milk (IFM) either entirely or in conjunction with breast milk during the first four months after birth. The presence of the emerging pathogen Enterobacter sakazakii in IFM has been associated with rare but fatal cases of neonatal infections and deaths. There is thus a need for accurate methods for the rapid detection of E. sakazakii in foods. At present, the methods used to detect and identify this micro-organism are inadequate, controversial and contradictory. The aim of this study was to determine the most suitable method for E. sakazakii detection after evaluation of the currently available methods. A further aim was to optimise a polymerase chain reaction (PCR) method for the detection of only viable E. sakazakii cells utilising the DNA-intercalating dyes ethidium monoazide (EMA) and propidium monoazide (PMA). The Food and Drug Administration (FDA) method for E. sakazakii detection was utilised to select 50 isolates from IFM and 14 from the environment, regardless of colony appearance. These isolates were identified by sequencing a 1.5 kilobase (kb) fragment of the 16S ribosomal DNA (rDNA) and by using the National Centre for Biotechnological Information (NCBI) database to confirm the closet known relatives. Seven of the 50 (14%) IFM isolates and six of the 14 (43%) environmental isolates were identified as E. sakazakii. The methods that were evaluated for accuracy in detecting and identifying these E. sakazakii isolates included yellow pigment production on tryptone soy agar (TSA), chromogenic Druggan-Forsythe-Iversen (DFI) and Enterobacter sakazakii (ES) agars and PCR using six different species-specific primer pairs described in the literature. The suitability of the FDA method was lowered by the low sensitivity, specificity and accuracy (87%, 71% and 74%, respectively) of using yellow pigment production for E. sakazakii identification. DFI and ES agars were shown to be sensitive, specific and accurate (100%, 98% and 98%, respectively) for the detection of E. sakazakii. The specificity of the PCR amplifications was found to vary between 8% and 92%, with Esakf and Esakr being the most accurate of the primer pairs evaluated. The current FDA method for E. sakazakii detection requires revision in the light of the availability of more sensitive, specific and accurate detection methods. Based on the results obtained in this study, a new method is proposed for the detection of E. sakazakii in food and environmental samples. This proposed method replaces the culturing steps on violet red bile glucose agar (VRBGA) and TSA with culturing on chromogenic DFI or ES agar. For identification and confirmation of presumptive E. sakazakii isolates, the oxidase test, yellow pigment production and API biochemical profiling is replaced by DNA sequencing and/or species-specific PCR with the most accurate primer pair (Esakf and Esakr). The amendments to the current FDA method will reduce the time to detect E. sakazakii from approximately 7 days to 4 days and should prove to be more sensitive, specific and accurate for E. sakazakii detection. In this study, a novel PCR-based method was developed which was shown to be capable of discriminating between viable and dead E. sakazakii cells. This was achieved utilising the irreversible binding of bacterial DNA to photo-activated PMA or EMA in order to prevent PCR amplification from the dead cells. At concentrations of 50 and 100 μg.ml-1, PMA completely inhibited PCR amplification from dead cells, while causing no significant inhibition of the PCR amplification from viable cells. EMA was equally effective in preventing PCR amplification from dead cells, however, it also inhibited PCR amplification from viable cells. PMA-PCR in particular, will be useful for assessing the efficacy of processing techniques, as well as for monitoring the resistance, survival strategies and stress responses of E. sakazakii. This will be an important step in the efforts to eliminate E. sakazakii from food and food production environments. / AFRIKAANSE OPSOMMING: Die Wêreld Gesondheidsorganisasie (WGO) beraam dat ten minste 75% van alle babas net baba formule melk (BFM) of BFM in kombinasie met moedersmelk in die eerste vier maande na geboorte kry. Die teenwoordigheid van die voortkomende patogeen Enterobacter sakazakii in BFM is al geassosieer met skaars maar noodlottige gevalle van neonatale infeksies en sterftes. Akkurate metodes word dus benodig vir die vinnige deteksie van E. sakazakii in voedsel. Die metodes wat huidiglik gebruik word vir die deteksie en identifikasie van hierdie mikroörganisme is onvoldoende, kontroversieël en teenstrydig. Die doel van hierdie studie was om die beste metode vir die deteksie van E. sakazakii te bepaal, na 'n evaluasie van die metodes wat huidiglik beskikbaar is. 'n Verdere doel was om 'n polimerase ketting reaksie (PKR) metode vir die deteksie van slegs lewensvatbare E. sakazakii selle te optimiseer deur gebruik te maak van die DNSbindende kleurstowwe, etidium mono-asied (EMA) en propidium mono-asied (PMA). Die Voedsel en Medisyne Administrasie (VMA) se metode vir E. sakazakii deteksie is gebruik om, ongeag van die kolonie kleur, 50 isolate vanuit BFM en 14 isolate vanuit die omgewing te kies. Hierdie isolate is geïdentifiseer deur die DNS volgorde van 'n 1.5 kilo-basis (kb) fragment van die 16S ribosomale DNS (rDNS) te bepaal en die Nationale Sentrum vir Biotegnologiese Informasie (NSBI) databasis te gebruik om die mees verwante spesie te bevestig. Sewe van die 50 (14%) BFM isolate en ses van die 14 (43%) omgewings isolate is geïdentifiseer as E. sakazakii. Die metodes wat geëvalueer is in terme van akkuraatheid vir deteksie en identifikasie van hierdie E. sakazakii isolate het PKR met ses verskillende spesie-spesifieke peiler pare soos beskryf in die literatuur, geel-pigment produksie op triptoon soja agar (TSA) en chromogeniese Druggan-Forsythe-Iversen (DFI) en Enterobacter sakazakii (ES) agars ingesluit. Die geskiktheid van die VMA metode is verlaag deur die lae sensitiwiteit, spesifisiteit en akkuraatheid (87%, 71% en 74% onderskeidelik) van geel pigment produksie vir E. sakazakii identifikasie. Chromogeniese DFI en ES agars was sensitief, spesifiek en akkuraat (100%, 98% en 98% onderskeidelik) vir die identifikasie van E. sakazakii. Die spesifisiteit van die PKR produkte het gewissel tussen 8% en 92%, en Esakf en Esakr is as die akkuraatste geëvalueerde peiler paar geidentifiseer. Die huidige VMA metode vir E. sakazakii deteksie vereis hersiening aangesien meer sensitiewe, spesifieke en akkurate deteksiemetodes voortdurend beskikbaar word. 'n Nuwe metode, gebaseer op die resultate van hierdie studie, word voorgestel vir die deteksie van E. sakazakii in voedsel- en omgewingsmonsters. Die voorgestelde metode vervang die kwekingsstap op violet rooi gal glukose agar (VRGGA) en TSA deur kweking op chromogeniese DFI of ES agars. Verder word die oksidase toets, geel pigment produksie en API biochemiese profiele van vermoeidelike E. sakazakii isolate vervang deur DNS volgorde bepaling en/of spesie-spesifieke PKR met die mees spesifieke peiler paar (Esakf and Esakf) vir die identifikasie en bevestiging van E. sakazakii. Die voorgestelde wysigings van die VMA metode sal die tydsduur van E. sakazakii identifikasie van 7 dae na 4 dae verminder, en behoort ook meer sensitief, spesifiek en akkuraat te wees vir die deteksie van E. sakazakii. 'n Nuwe PKR-gebaseerde metode wat tussen lewensvatbare en dooie E. sakazakii selle kan onderskei is in hierdie studie ontwikkel. Dit is bereik deur die onomkeerbare binding van bakteriële DNS aan lig-geaktiveerde EMA of PMA om die PKR amplifisering van dooie selle te voorkom. Konsentrasies van 50 en 100 μg.ml-1 PMA het PKR amplifikasie heeltemal geïnhibeer, terwyl geen inhibisie van lewensvatbare selle bespeur kon word nie. EMA was ook suksesvol in die voorkoming van die PKR amplifikasie van dooie selle, alhoewel daar ook 'n mate van DNS inhibisie was tydens die amplifikasie van lewensvatbare selle. PMA-PKR kan ook van nut wees vir die assessering van die doeltreffendheid van prosesseringstegnieke, en ook vir die waarneming van die weerstandigheid, oorlewingsstrategieë en stresresponse van E. sakazakii. Dit sal 'n belangrike stap wees in pogings om E. sakazakii van voedsel en voedsel produksieomgewings te elimineer.

Thesis (Msc Food Sc (Food Science))--University of Stellenbosch, 2006. / The food and beverage industry generates large volumes of wastewater annually. The disposal of factory effluent from the fruit processing industry has always been a cause of concern to both the fruit processors and controlling bodies responsible for effluent management. Traditional disposal of wastewater into sewerage works has become undesirable due to its economical and environmental impacts. Therefore, on-site anaerobic treatment of wastewater has received considerable interest due to lower capital outlays and energy recovery possibilities. Thus, the aim of this study was to establish an operational treatment profile for an anaerobic pond system treating fruit-processing wastewater. The specific activity of the microbial populations was also monitored to determine the effect of the fruit processing seasons (peak and off-peak season). The biogas production potential at various temperatures was also assessed to determine the viability of methane recovery. The influence of the processing and environmental conditions on the ponds’ performance was established by monitoring various process parameters. The results showed that the chemical oxygen demand (COD) levels decreased during the off-peak season but the pond pH remained relatively stable between 6.0 and 6.4 during the entire year. Pond alkalinity was found to be dependent on the regular lime dosing to maintain the necessary alkalinity. The volatile fatty acid (VFA) concentrations indicated that the microbial populations of the pond were functioning well. However, a decrease in microbial activity and VFA concentrations were observed at the lower temperatures during the winter months. The temperature profile of the pond showed that the pond temperature was impacted by the fluctuations in the ambient air temperature. The general trend established by the operational treatment profile clearly showed the impact of the peak and off-peak season. The sludge activity of the anaerobic pond was evaluated to determine the effect of the apple-processing peak and off-peak season on the specific activity of the acidogenic and methanogenic populations within the sludge. An activity test using four different test media was used during the activity assays. Sludge samples were taken at four different sampling positions across the pond’s sludge bed. The sludge was also subjected to a biogas formation study, which was designed to simulate pond conditions on laboratory scale in order to evaluate the biogas production potential of the anaerobic pond. The cumulative biogas volume and total CH4 composition showed little or no difference between the four sludge sampling sites. A major difference was found between the activity of the microbial populations during the peak and off-peak seasons. The overall trend regarding the biogas production rate (SB) and the methane production rate (SM) values showed an increased activity during peak-season and a decreased activity during off-peak season. For the biogas formation test the highest incubation temperature (25°C) resulted in the most biogas being produced, followed by 18°C, and with 10°C resulting in the lowest biogas volume. The biogas formation tests indicated that microbial activity and therefore biogas production was dependent on especially favourable temperature conditions. The pond and activity of the microbial populations are therefore influenced by factors like environmental changes such as decreased air temperatures and substrate changes such as decreased COD concentrations during the off-peak season. This in turn influences the rate of biogas production as well as the methane production rate. The theoretical CH4 calculations and estimates based on the results obtained during the biogas formation tests indicated that CH4 recovery from the anaerobic pond would definitely be a worthwhile consideration. If it were assumed that the estimated CH4 volumes (based on only 15% of the pond volume for practical reasons) obtained could be applied as an energy source, the minimum yearly savings in coal usage would amount to about R 665 000. This study was valuable in evaluating the factors such as pond conditions, pond activity and air temperatures and the effect on the biogas production potential as well as more importantly, CH4 production for the purpose of energy recovery.

Thesis (Msc Food Sc (Food Science))--University of Stellenbosch, 2007. / The distillery industry generates large volumes of heavily polluted wastewater and thus effective wastewater treatment is essential. It has been reported that a chemical oxygen demand (COD) reduction of more than 90% can be achieved when wine distillery wastewater (WDWW) is treated in an upflow anaerobic sludge blanket (UASB) reactor. The first objective of this study was to investigate UASB treatment of WDWW and to try to enhance the efficiency by using ozonation treatments. Secondly, the impact of grain distillery wastewater (GDWW) on UASB granules was determined. The third objective was to determine whether ozonation and enzymatic treatment combinations might improve the biodegradability of GDWW and thus make GDWW more amenable to UASB treatment. It was found that UASB treatment combined with ozonation improved the WDWW treatment efficiency. When diluted WDWW (chemical oxygen demand COD = 4 000 mg.L-1) was ozonated (dose = 47 mg.L-1) in a 50 L venturi circulating contactor system, the COD reduction was 7%. When WDWW was treated in a laboratory-scale UASB reactor (substrate pH = 7.0, COD = 4 000 mg.L-1 and organic loading rate (OLR) = 4.0 kg COD.m-3.d-1), the COD reduction was 92%. When the UASB treatment was combined with either pre- or postozonation, the COD reduction was 94 and 96%, respectively. When UASB treatment was combined with pre- and post-ozonation, a COD reduction of 98% was achieved. The activity of the UASB granules was also found to improve over time, despite the addition of the ozonation treatment. It has been reported that operational problems occur when GDWW is treated in an UASB reactor as a result of the encapsulation of the granules. This was confirmed when granules from a full-scale UASB treating WDWW became encapsulated in a layer after being exposed to GDWW (COD = 4 000 mg.L-1) for 24 d. The results showed that the lipid content of the granules increased from 1.25 to 60.35 mg lipid.g-1 granule over the 24 d exposure period. Therefore, granules exposed to GDWW were encapsulated in a lipid-rich layer and as a result the contact between the GDWW and microbial consortium in the granules was reduced. The operational problems found during the industrial UASB treatment of GDWW were ascribed to the encapsulation of the granules. Combinations of ozonation (dose = 1 476 mg.L-1) generated in a 2 L bubble column and enzymatic treatments (1% FogFreeTM (FF) dosage and 2 d incubation at 35°C) were found to improve the biodegradability of GDWW. This improvement was in terms of lipid reduction in GDWW, granule activity and visual appearance of the encapsulating layer of the granules. The highest lipid reduction (90%), highest granule activity, lowest lipid content of the granules (3.74 ± 0.10 mg.g-1 granule) and best visual appearance were achieved in ozonated GDWW treated with 1% FF, followed by just ozonation. The higher lipid reduction and subsequent higher granule activity were ascribed to the reduction in lipids which resulted in the fact that fewer lipids were available to encapsulate the granules. As a result of the lipid reduction, the granule activity improved and the GDWW was made more amenable to UASB treatment. This study proved that UASB treatment combined with ozonation led to an enhancement of the treatment efficiency of WDWW. It was also found that the cause of the operational problems during UASB treatment of GDWW was as a result of the granules being encapsulated in a lipid-rich layer. It was established that treating GDWW prior to UASB treatment improved the biodegradability of GDWW. The data from the study showed that high lipid reduction in the GDWW directly led to better granule activity, lower granule lipid content and a thinner encapsulating layer. Based on the data from this study, it is recommended that GDWW be ozonated prior to other treatments because it can be done inline and the costs would be lower than that of enzymatic treatments.

Thesis (MSc Food Sc (Food Science))--University of Stellenbosch, 2010. / ENGLISH ABSTRACT: This study focussed on the sensory effects of the main volatile compounds produced by Brettanomyces yeast causing spoilage in wine. This research firstly aimed to determine the detection thresholds of eight Brett-related spoilage compounds in wine. The second aim was to determine the sensory effect of the four most important Brett-related compounds when present individually in wine. The third aim was to determine the sensory effects of these four compounds when present in wine in a range of combinations, and to further investigate their effect on consumer liking. Finally, this project aimed to investigate the incidence of these compounds in a small range of South African wines. The sensory detection thresholds of 4-ethylphenol, 4-ethylguaiacol, 4-ethylcatechol, 4- vinylphenol, 4-vinylguaiacol, isovaleric acid, isobutyric acid and acetic acid were determined. Apart from 4-ethylcatechol, these values generally agreed well with recent literature where values determined in wine are available. However, the discrepancies highlighted the importance of the effect of the medium (wine) when determining sensory detection thresholds. The use of the median as alternative calculation method was also investigated, and it was found that this method gives more insightful results than the standard American Society of Testing Materials (ASTM E679-04) method. Four compounds, namely 4-ethylphenol, 4-ethylguaiacol, 4-ethylcatechol and isovaleric acid were profiled individually in wine using a trained sensory panel. It was found that all four compounds caused a suppression of the natural berry-like character in the wine, which induced a sick-sweet character. 4-ethylphenol contributed Elastoplast™ and leather aromas in the wine, both of which are commonly associated with Brettanomyces taint. 4-ethylguaiacol added a medicinal aroma to the wine, and 4-ethylcatechol and isovaleric acid were responsible for savoury and pungent aromas, respectively. 4-ethylphenol, 4-ethylguaiacol, 4-ethylcatechol and isovaleric acid were also profiled in combination according to the central composite design. Several univariate and multivariate methods were applied to the dataset obtained. PARAFAC, a multiway method not widely utilized regarding sensory data, was applied to the data, the results of which were complementary to those obtained during univariate and multivariate analyses. It was found that there is a great deal of interaction between the four compounds profiled in terms of sensory effects. The most notable was the Elastoplast™ attribute, the intensity of which was affected by all four compounds. The pungent attribute was also affected by the 4-ethylphenol concentration. Consumer analysis revealed that some of the samples spiked with Brettanomyces-spoilage compounds were preferred to the unspiked (control sample). However, no further relationship could be found between consumer liking and either chemical composition or sensory profile. It is therefore speculated that consumer liking of Brettanomyces infected wine is driven by more complex sensory or socio-demographic factors. Finally, the concentration of 4-ethylphenol, 4-ethylguaiacol, 4-ethylcatechol, 4-vinylphenol, 4- vinylguaiacol, isovaleric acid, isobutyric acid and acetic acid was determined in a small set of South African wines, selected to contain a high proportion of wines spoiled by Brettanomyces. Significant correlations were found between 4-ethylphenol and 4-ethylguaiacol, as well as 4- ethylphenol and isovaleric acid. However, no correlation could be found between 4-ethylphenol and 4-ethylcatechol. It is speculated that this lack of relationship is due to the different precursor profiles present in the analysed wines. This study paved the way for future investigations on the sensory effects of Brettanomyces spoilage in Pinotage red wine. / AFRIKAANSE OPSOMMING: Hierdie studie het gefokus op die sensoriese invloed van die belangrikste vlugtige komponente wat deur die Brettanomyces gis geproduseer word en bederf veroorsaak in wyn. Eerstens is gefokus op die bepaling van die deteksiedrempelwaardes van agt Brett-verwante bederwende komponente. Die tweede doelwit was om die sensoriese invloed van vier van die mees belangrike Brett-komponente te bepaal wanneer hulle individueel in wyn voorkom. Die derde doelwit was om die sensoriese invloed van hierdie vier komponente te bepaal wanneer hulle in verskillende kombinasies in wyn voorkom, asook die effek daarvan op verbruikervoorkeur. Laastens is gepoog om die voorkoms van hierdie komponente in ‘n klein seleksie van Suid- Afrikaanse wyne te bepaal. Die sensoriese deteksiedrempelwaardes vir 4-etielfenol, 4-etielguaiacol, 4-etielcatechol, 4- vinielfenol, 4-vinielguaiacol, isovaleraatsuur, isobuteraatsuur en asynsuur is bepaal. Met die uitsondering van 4-etielcatechol het die waardes oor die algemeen goed ooreengestem met waardes wat onlangs in die wetenskaplike literatuur gepubliseer is. Die uitsonderings het egter die belangrikheid van die medium (wyn) gedurende die bepaling van sensoriese deteksiedrempelwaardes uitgelig. Die gebruik van die mediaan as ‘n alternatiewe berekeningsmetode is ook ondersoek en daar is gevind dat hierdie metode meer insiggewende resultate lewer as die standaard American Society of Testing Materials (ASTM E679-04) metode. Vier komponente naamlik 4-etielfenol, 4-etielguaiacol, 4-etielcatechol en isovaleraatsuur is individueel in wyn geprofileer met behulp van ‘n opgeleide sensoriese paneel. Daar is gevind dat al vier die komponente die natuurlike bessiekarakter in die wyn onderdruk terwyl dit aanleiding gee tot ‘n onnatuurlike soet karakter. 4-etielfenol is gekenmerk aan Elastoplast™ en leeragtige aromas in die wyn en beide van hulle word algemeen geassosieer met Brettanomyces bederf. 4-etielguaiacol het ‘n medisinale aroma tot die wyn toegevoeg en 4- etielcatechol en isovaleraatsuur het respektiewelik souterige (“savoury”) en sterk (“pungent”) aromas tot gevolg gehad. 4-etielfenol, 4-etielguaiacol, 4-etielcatechol en isovaleraatsuur is ook in verskeie kombinasies geprofileer volgens die sentrale saamgestelde ontwerp (“central composite design”). Verskeie enkelveranderlike en meerveranderlike statistiese analisemetodes is ook op die datastel uitgevoer. PARAFAC, ‘n meerrigtingsmetode wat nie normaalweg vir sensoriese analise data gebruik word nie, is ook uitgevoer op die data en die resultate was komplimentêr tot die van die enkelveranderlike en meerveranderlike analisemetodes. Daar is gevind dat, met betrekking tot sensoriese effekte, daar noemenswaardige interaksie tussen die vier komponente plaasvind. Die mees opmerklike hiervan was die Elastoplast™ aroma, waarvan die intensiteit deur al vier die ander komponente geaffekteer is. Verder is die sterk (“pungent”) aroma beïnvloed deur die 4-etielfenol konsentrasie. Verbruikersvoorkeur-analise het aangedui dat sommige van die monsters waarby Brettanomyces bederwende komponente gevoeg is, verkies word bó die kontrole-wyn. Daar kon egter geen verdere verband gevind word tussen die verbruiker se voorkeur en, nog die chemise komposisie of sensoriese profiele, van die wyn nie. Daar kan dus gespekuleer word dat verbruiker voorkeur van Brettanomyces bederfde wyn gedryf word deur meer komplekse en sosio-demografiese faktore. Laastens is die konsentrasies van 4-etielfenol, 4-etielguaiacol, 4-etielcatechol, 4-vinielfenol, 4- vinielguaiacol, isovaleraatsuur, isobuteraatsuur en asynsuur in ‘n seleksie van Suid-Afrikaanse wyne bepaal. Dié wyne is spesifiek so gekies sodat ‘n aansienlike aantal van hulle met Brettanomyces bederf was. Betekenisvolle korrelasies is gevind tussen 4-etielfenol and 4- etielguaiacol, sowel as 4-etielfenol en isovaleraatsuur. Daar is egter geen korrelasie tussen 4- etielfenol and 4-etielcatechol gevind nie. Daar word vermoed dat hierdie gebrek aan korrelasie te wyte is aan die voorloperkomponent profiele teenwoordig in die wyne. Hierdie studie het die weg gebaan vir verdere ondersoeke na die sensoriese effekte van Brettanomyces bederf in Pinotage rooi wyn.

Thesis (MSc Food Sc )--Stellenbosch University, 2004. / ENGLISH ABSTRACT: The South African apple juice processing industry is growing rapidly and during the harvesting season the wastewater volumes and organic loads increase significantly with a considerable environmental impact. These larger apple juice processing wastewater (AJPWW) volumes and chemical oxygen demand (COD) loads subsequently lead to faster increases in the organic loading rate (OLR) of an upflow anaerobic sludge bed (UASB) wastewater treatment system and it is necessary to know if the treatment system can handle such drastic increases over short periods. The objective of the study were to evaluate the efficiency of the UASB process in the treatment of an AJPWW; to determine what effect a substrate viscosity increase, based on a pectin calcium gel has on the performance of an UASB system, and to determine what impact ozonation has on the pectin content, gelformation ability and biodegradability of the AJPWW. The ability of the UASB to maintain stability during the apple-processing season was investigated by increasing the OLR from 2.9 to over 14.0 kg COD.m-3.d-1 in 131 days. During this time the COD removal remained constant at 85%, while the pH and alkalinity remained at levels indicative of good reactor stability. It was thus concluded that the UASB reactor could operate successfully during the apple-harvesting season when wastewater volumes and organic loads increase significantly. In the study it was found that the viscosity of the AJPWW, containing 750 mq.L-1 pectin, increased from 8.5 to 47.0 cps after a 312 rnq.L-1 Ca2 + addition. This increased viscosity substrate was then fed to an UASB reactor at an OLR of 15.0 kg COD.m-3.d-1. During a 12 day increased viscosity (47 cps) feeding stage the COD removal decreased from 94 to 11%, while the reactor pH decreased from 7.5 to 4.9. During this period, pectin accumulated in the UASB and led to biomass washout and rapid UASB failure. The possible elimination of pectin by ozonation was thus investigated, and a 77% decrease in pectin content and 76% decrease in gel formation ability occurred after ozonation. The effect of pre- and post-ozonation on the efficiency of the UASB system was subsequently investigated. It was found that a 10 min pre-ozonation decreased the AJPWW COD by 19% and the total suspended content by 36%, while the soluble portion of the total COD was increased from 81.7 to 92.4%. This increase in soluble COD content should lead to increased wastewater biodegradability. The ozonated AJPWW was then used to replaced the raw non-ozonated AJPWW as reactor feed. Results showed that the COD removal increased from 78 to 90% within 24 h of starting with the ozonated feed. It was also found that the reactor stability improved after AJPWW pre-ozonation as an OLR increase from 10.0 to 16.6 kg COD.m-3.d-1 in 23 days did not detrimentally influence the stability of the reactor. This reactor effluent (COD = 465 rnq.L-1) was then post-ozonated which resulted in 64.8% COD and 79.0% colour reductions. The final effluent had a COD of 180 rnq.L-1 (98% reduction). The ability of the ozonation/digestion system as described in this study to degrade AJPWW at a higher OLR is of value to the apple industry, as it may lead to larger organic pollutant removals and thus a more effiecient treatment system. Increased reactor performance will directly improve the quality of the final wastewater produced, which in turn will have a significant impact on the treatment ability of the South African apple processing industry currently limited by the production of large wastewater volumes. / AFRIKAANSE OPSOMMING: Gedurende die oesseisoen word groot volumes afvalwater met 'n hoe organiese lading in die vinnig groeiende Suid-Afrikaanse appelsapprosesseringsbedryf geproduseer. Dit het 'n groot impak op die omgewing. Die groter volumes appelsapprosesseringsafvalwater (ASPAW) met 'n hoë organiese lading het 'n vinniger verhoging in organiese ladingstempo's (OLT) van 'n UASB-waterbehandelingstelsel tot gevolg. Daarom is dit belangrik om te weet of die stelsel die drastiese verhoging oor kort tydperke kan hanteer. Die doel van hierdie studie was die evaluering van die effektiwiteit van die UASB-proses in die behandeling van ASPAW; om te bepaal watter effek 'n substraatviskositeitsverhoging, gebaseer op 'n pektien-kalsium-jel, op die doeltreffendheid van 'n UASB-stelsel het; en om te bepaal watter impak osonering op die pektieninhoud, jelvormingsvermoe en bioafbreekbaarheid van ASPAW het. Die vermoe van die UASB om stabiliteit te handhaaf gedurende die appelsapprosesseringseisoen is ondersoek deur die OLT van 2,9 tot bo 14,0 kg CSB.m-3.d-1 te verhoog oor 131 dae. Gedurende hierdie tyd het die chemiese suurstofbehoefte- (CSB-) verwydering konstant gebly by 85%, terwyl die pH en alkaliniteit ook op vlakke aanduidend van goeie reaktorstabiliteit gebly het. Daar is sodoende bewys dat die UASB-reaktor suksesvol kan presteer tydens die appelsapprosesseringseisoen, wanneer daar 'n beduidende verhoging in OLT plaasvind. In die studie is daar gevind dat die viskositeit van die ASPAW, wat 750 mq.L-1 pektien bevat, van 8,5 tot 47,0 cps toeneem na die byvoeging van 312 rnq.L-1 Ca2+. Hierdie verhoogde vikositeitsubstraat is tot die UASB-reaktor toegevoeg teen 'n OLT van 15,0 kg CSB.m-1.d-1. Gedurende 'n 12-dae toevoer van verhoogde viskositeit (47 cps), het die CSB-verwydering van die reaktor afgeneem van 94% na 11%, terwyl die pH gedaal het van 7,5 na 4,9. Gedurende hierdie tydperk het pektien in die UASB geakkumuleer, wat gelei het tot die uitspoel van biomassa en vinnige UASB-reaktormislukking. Die moontlike eliminasie van pektien, deur osonering, is daarom ondersoek. 'n Verlaging van 77% in pektieninhoud en 76% in jelvormingsvermoe het na osonering plaasgevind. Die effek van pre- en post-osonering op die effektiwiteit van 'n UASB-stelsel is gevolglik ondersoek. Daar is gevind dat 'n 10 minute pre-osonering die CSB van die ASPAW met 19% verlaag en die totale inhoud van gesuspendeerde vaste stowwe met 36% verlaag, terwyl die oplosbare gedeelte van die totale CSB van 81,7% tot 92,4% gestyg het. Die verhoging in oplosbare CSB-inhoud behoort tot verhoogde bioafbreekbaarheid van ASPAW te lei. Die geosoneerde ASPAW is gebruik om die rou, ongeosoneerde ASPAW as reaktorsubstraat te vervang. Die resultate het getoon dat die CSB-verwydering verhoog het van 78% na 90% na 'n 24-uur toevoer van geosoneerde substraat. Daar is ook gevind dat die reaktorstabiliteit toegeneem het na ASPAW osoneering, aangesien 'n OLT-verhoging van 10,0 na 16,6 kg.CSB.m-3.d-1 in 23 dae nie die stabiliteit van die reaktor nadelig beinvloed het nie. Hierdie reaktoruitvloeisel (CSB = 465 rnq.L-1) is hierna gepost-osoneer, wat 'n 64,8% CSB- en 79,0% kleurverlaging tot gevolg gehad. Die finale uitvloeisel het 'n CBS-inhoud van 180 rnq.L-1 gehad (98,1% verwydering). Die vermoe van die osonering-/verteringstelsel om ASPAW te degradeer teen 'n hoër OLT, soos beskryf in hierdie studie, is van waarde tot die appelsapprosesseringsbedryf, aangesien dit tot groter organiese afvalstofverwydering kan lei en dus 'n meer effektiewe behandelingstelsel tot gevolg kan hê. Verhoogde reaktordoeltreffendheid sal 'n direkte verbetering tot gevolg hê in die gehalte van die finale afvalwater wat geproduseer word, wat op sy beurt 'n beduidende impak sal hê op die behandelingsvermoe van die appelsapprosesseringsbedryf, wat tans beperk word deur die produksie van groot volumes afvalwater.

Thesis (MSc Food Sc )--Stellenbosch University, 2002. / ENGLISH ABSTRACT: South Africa is a developing country that relies heavily on its agricultural sector for economical welfare especially in the Western Cape Province. However, development gives rise to new technologies, new products, economical stability and unfortunately also to the production of larger volumes of liquid and solid waste. Anaerobic composting is becoming a very attractive treatment option for solid waste disposal because of its unique operational advantages and two valueadded by-products, compost and biogas. Over the last decade progress has been made in anaerobic digestion of solid wastes, but no literature could be found on the anaerobic composting of apple and peach pomace. The main objective of this study was to develop a method to anaerobically compost apple and peach pomace. In the first phase important operational parameters were identified and a method was developed to optimise the parameters. In the second phase of the study, the scaling-up and optimisation of the process were the major objectives. During the first phase of this research 2 L modified glass containers were used as composting units. The most important operational parameters (leachate pH, inoculum source and size, and initial moisture levels) were identified. Anaerobic compost from previous tests, brewery granules and anaerobic sludge were also used as inocula and evaluated for the best source of microbes. After optimising all the identified parameters, good results were obtained, which included higher biogas production, good volume reductions, less bad aromas and a compost product with a neutral pH. After developing the 2 L laboratory-scale method to compost the apple pomace anaerobically, the next step was to ascertain if the method would work if larger volumes of solid fruit waste were composted. A special 20 L composting unit made of PVC was designed to suit the operational requirements of the anaerobic composting process. It was also decided to mix apple pomace and peach pulp together and to use this solid waste source as part of the composting substrate. Different inocula, including cattle manure, anaerobic sludge, brewery granules and anaerobic compost produced in the previous tests, were used. Although good results were obtained with the anaerobic compost and cattle manure as inoculum, the aim was also to decrease the composting period by shortening the pH stabilisation period. To achieve this, it was decided to add NaHC03 to the substrate to be composted to facilitate a faster pH stabilisation. The composting period was subsequently shortened to 25 days with satisfactory results, which included a volume reduction, biogas production and faster pH stabilisation. An upflow anaerobic sludge blanket (UASB) bioreactor was also used to assist the composting process by facilitating the removal of the VFA's present in the composting leachate. This proved to be a valuable addition to the composting process as the UASB bioreactor also provided the composting units with a 'moisturising liquid', which was 'enriched' with a consortium of active anaerobic bacteria when the effluent from the bioreactor was re-added to the composting units. With all the operational parameters in place, good results were obtained and these included a volume reduction of 60% (m/m), a good biogas production, a composting period of only 25 days, a compost that was free of bad aromas, a final compost pH of > 6.5, final leachate COD values of less than 3 000 rnq.l", and a final leachate VFA's concentration of between 0 and 250 rnq.l". If in future research further scaling-up is to be considered, it is recommended that the composting unit be coupled directly to the UASB bioreactor, thus making the process continuous and more practical to operate. If the operational period of the anaerobic composting set-up could be further shortened and the inoculum adapted so that the process could be used for the treatment of other difficult types of solid wastes, it would probably be advantageous for the fruit processing industry to use this method as an environmental control technology. / AFRIKAANSE OPSOMMING: Suid-Afrika is 'n ontwikkelende land wat baie afhanklik is van die sukses van die landbousektor vir ekonomiese welstand, veral in die Wes Kaap Provinsie. Ontwikkeling gaan gepaard met nuwe tegnologie, nuwe produkte, ekonomiese stabiliteit en daarmee saam gaan die produksie van groter volumes vlooiebare en soliede afvalprodukte. Anaërobiese kompostering is tans besig om opgang te maak as en doeltreffende behandelingstegnologie vir vaste afvalstowwe. Tydens die laaste dekade is baie vooruitgang gemaak in die veld van anaërobiese vertering asook kompostering van afvalmateriaal met en hoë vaste stof inhoud. Anaërobiese kompostering van appel- en perskepulp, afkomstig van die versappingsindustrie, het tot dusver min aandag geniet. Die hoofdoel van hierdie navorsing was om 'n anaërobiese komposterings metode te ontwikkel vir die beheer van vrugte afval om sodoende die basis neer te lê vir en nuwe tegnologie wat baie voordele (biogas en kompos) inhou. In die eerste fase is die belangrikste operationele parameters geïdentifiseer om sodoende beter beheer oor die anaërobiese proses uit te oefen. In die tweede fase is die anaërobiese proses wat gedurende die eerste fase ontwikkel is, opgeskaal om optimum resultate te verkry. Gedurende die eerste fase van hierdie verhandeling was 2 L gemodifiseerde glas houers gebruik as komposteringseenhede. Die belangrikste operasionele parameters (pH beheer, inokulasie grootte, vloeistofvlakke en hoeveelheid vog asook vlugtige vetsuur produksie en verwydering) vir die beheer van die anaërobiese komposteringsproses was geïdentifiseer en gebruik as uitgangspunt om 'n anaërobiese komposteringsmetode te ontwikkel. Anaërobiese slyk, brouery granules en anaërobiese kompos van vorige eksperimente was as inokula gebruik. Gedurende hierdie studies was goeie resultate verkry en het 'n hoë biogas produksie, goeie volume reduksies, vermindering van slegte aromas en kompos met 'n neutrale pH ingesluit. . Nadat hierdie goeie resultate met die 2 L laboratorium-skaal metode verkry was, was groter volumes vaste vrugte afval gebruik om te bepaal of dieselfde metode toegepas kan word op en groter skaal. Spesiale 20 L komposteringseenhede was ontwerp om aan die operasionele vereistes van 'n anaërobiese proses te voldoen. Dit was ook besluit om appel pulp met perske pulp te meng en te gebruik as deel van die komposteringssubstraat. Verskeie inokula was weereens gebruik en het die volgende ingesluit: vars beesmis, anaërobiese slyk, brouery granules en anaërobiese kompos van vorige eksperimente. Hoewel baie goeie resultate met vars beesmis en anaërobiese kompos as inokula verkry was, was 'n volgende doel gewees om die kompoterings tydperk te verkort deur die pH vinniger te stabiliseer. Daar was besluit om NaHC03 by die komposteringssubstraat te voeg en so 'n vinniger pH stabilisasie te fasiliteer. 'n UASB ('upflow anaerobic sludge blanket') bioreaktor was ook gebruik om die komposteringsproses aan te help deur die vlugtige vetsure wat in die kompostloog teenwoordig was, te verwyder. Die insluiting van die bioreaktor in die anaërobiese komposteringsproses het bygedra tot die sukses van die proses deurdat die uitvloeisel as 'n vogmiddel vir die komposteringseenhede gebruik was en 'n konsortium van aktiewe anaërobiese bakterieë bevat het. Nadat al die operationele parameters in plek was, was goeie resultate bereik en het die volgende ingesluit: 'n volume reduksie van 60% (m/m), goeie biogas produksie, 'n komposteringstyd van 25 dae, 'n kompos wat vry was van slegste aromas, 'n finale kompos pH van >6.5, finale loog CSB van <3 000 rnq.l' en 'n finale vetsuur konsentrasie van tussen 0 en 250 mq.l'. lndien verdere navorsing onderneem word, word dit aanbeveel dat die UASB bioreaktor direk aan die komposteringseenheid gekoppel word om sodoende die proses meer aaneenlopend en die proses prakties makliker uitvoerbaar te maak. Indien die operationele tydperk nog korter gemaak kan word en die inokulum aanpasbaar kan wees om moeilik verteerbare afvalprodukte te akkomodeer, sal hierdie tegnologie baie voordelig wees as 'n metode om omgewingsbesoedeling te beheer

Thesis (MSc Food Sc )--Stellenbosch University, 2003. / ENGLISH ABSTRACT: Kepi is a refreshing, fermented dairy beverage that differs from other fermented milk products in that it is produced with a mixed microbial community which is confined to discrete grains. These grains can be recovered as a solid matrix at the end of the fermentation and then be reutilised as a starter to ferment the next batch of milk. The grain microbial community consists of a symbiotic association of yeasts and lactic acid bacteria, but the overall composition of the grains has not been completely elucidated. The microbes in the grains are embedded in a protein-polysaccharide Kefiran matrix, which appears essential for grain formation. The mechanism of grain formation is still not fully understood and it thus remains undecided which organism is really responsible for the production of this proteinpolysaccharide matrix. The aim of this study was to isolate, characterise and identify the microbes present in Kefiran from mass cultured South African grains and then to evaluate grain formation with these purified cultures isolated from Kefiran strings using a mass cultivation process. Sixteen strains of lactic acid bacteria and one yeast strain were isolated from Kefiran strings produced during the mass cultivation of South African Kepi grains. API technology, numerical clustering and DNA sequence comparisons were used to identify the purified isolates. The isolates were grouped into seven clusters by numerical clustering and clustering distance from selected reference and marker strains. The heterofermentative lactobacilli were identified as Lactobacillus parakefiri and Lb. kefiri and the homofermentative strains as Lb. delbrueckii ssp. bulgaricus, Lb. gallina rum, Lb. acidophilus and Lb. bavaricus. One isolate was found to be a member of the genus Lactobacillus, but was not positively identified to species level. Cultures isolated from Kefiran were evaluated for ability to grain formation by adding 1 x 109 cfu.ml:' bacteria and 1 x 108 cfu.ml' yeast to double pasteurised, full cream milk during the mass cultivation process. It was found that the control and all the cultures in double pasteurised milk showed grain accumulation indicating that other microbes were present in pasteurised and double pasteurised milk which had an influence on the grain forming ability. The cultures isolated from pasteurised and double pasteurised milk included members of the species Pediococcus, Acinetobacter, Lactococcus laetis ssp. lactis, Candida lipolytica, C. guilliermondii, Chryseobacterium meningosepticum, Pseudomonas putida and four isolates of the Bacillus cereus group. It was found that these rod-shaped "milk isolates" resulted in grain accumulation when inoculated into UHT milk and it was concluded that the "milk isolates" did contribute to grain formation. These isolates were then combined with the Kefiran cultures and this resulted in grains very similar to the traditional Kepi grains. These grains were made from Lb. gallinarum in double pasteurised milk as well with a combination of Lb. gallinarum, Lb. acidophilus, Lb. kefiri, Lb. delbrueckii ssp. bulgaricus, Candida lambica and Pseudomonas putida in URT milk. The grains were firm, elastic and did not dissolve in water but kept their structure and were retained when sieved. An acceptable Kepi beverage was produced from these grains. From these typically traditional grain characteristics it was concluded that, even though the microbial compositions were probably not the same, the general appearance was similar to traditional grains and that it is thus possible to produce grains from pure single strain Kefiran cultures and "milk isolates". Furthermore, it was possible to produce a Kepilike beverage from these grains, which included similar characteristics as the traditional Kepi beverage. / AFRIKAANSE OPSOMMING: Kepi is "n verfrissende, gefermenteerde suiweldrankie wat van ander gefermenteerde produkte verskil in die opsig dat dit vervaardig word deur Kepi korrels in melk te inkubeer. Die Kepi korrels kan aan die einde van die fermentasie herwin word en weer gebruik word om die volgende lot melk te fermenteer. Die korrels bestaan uit "n simbiotiese samestelling van giste en melksuurbakterieë, maar die presiese samestelling van die korrels is steeds onbekend. Die mikro-organismes is vasgevang in "n proteïen-polisakkaried Kefiran matriks en die Kefiran word as essensieel beskou vir korrelvorming. Die meganisme van korrelvorming bly steeds onbekend en daar is nog nie tot "n gevolgtrekking gekom oor watter organisme die Kefiran produseerder is nie. Die doel van die studie was om die mikro-organismes in Kefiran te isoleer en te identifiseer deur Suid-Afrikaanse Kepi korrels te massa kweek. Hierdie mikroorganismes was dan verder geëvalueer ten opsigte van korrel vorming. Sestien melksuurbakterieë isolate en een gis isolaat is geïsoleer vanuit die Kefiran. API tegnologie, numeriese groepering en DNA volgorde vergelykings was gebruik om die isolate te identifiseer. Die isolate is in sewe groepe verdeel volgens numeriese groepering. Die afstand van verwysings en merker organismes is ook in ag geneem. Die heterofermentatiewe organismes is geïdentifiseer as Lactobacillus parakefiri en Lb. kefiri en die heterofermentatiewe organismes as Lb. delbrueckii ssp. bulgaricus, Lb. gallina rum, Lb. acidophilus en Lb. bavaricus. Een isolaat kon nie geïdentifiseer word tot op spesie vlak nie, maar is verwant aan die genus Lactobacillus. Hierdie geïsoleerde Kefiran kulture is geëvalueer ten op sigte van korrelvorming, deur 1 x 109 kve.ml' van die bakterieë en 1 x 108 kve.ml' van die gis by dubbel gepasteuriseerde volroom melk te voeg tydens die massakwekings proses. Die kontrole wat geen bygevoegde kulture bevat nie, sowel as die wat wel bygevoegde kulture bevat, het korrel vorming getoon. Laasgenoemde toon dat daar organismes teenwoordig is in gepasteuriseerde en dubbel gepasteuriseerde melk wat "n rol kan speel tydens korrelvorming. Die kulture wat geïsoleer is vanuit gepasteuriseerde en dubbel gepasteuriseerde melk, sluit in: Pediococcus, Acinetobacter, Lactococcus laetis ssp. lactis, Candida lipolytica, C. guilliennondii, Chryseobacterium menigosepticum, Pseudomonas putida en vier isolate van die Bacillus cereus groep. Hierdie organismes wat uit melk geïsoleer is, het korrelvorming getoon in UHT melk en die gevolgtrekking kan gemaak word dat die "melk organismes" wel "n rol speel tydens korrel vorming. Hierdie "melk isolate" in kombinasie met die Kefiran kulture het korrels tot gevolg gehad wat baie dieselfde was as tradisionele Kepi korrels. Laasgenoemde korrels is gemaak deur Lb. gallina rum in dubbel gepasteuriseerde melk, sowel as deur "n kombinasie van Lb. gallina rum, Lb. acidophilus, Lb. kefiri, Lb. delbrueckii ssp. bulgaricus, Candida lambica en Pseudomonas putida in UHT melk. Die korrels was stewig, elasties, het nie opgelos in water nie en het hulle struktuur behou wanneer gesif. Wanneer hierdie tipiese tradisionele korrels se eienskappe in ag geneem word, kan die gevolgtrekking gemaak word dat alhoewel die mikrobiese samestelling van die korrels nie dieselfde is as die tradisionele korrel nie, is die algemene voorkoms en eienskappe dieselfde en dat dit wel moontlik is om korrels te produseer deur isolate geïsoleer vanuit Kefiran en melk. Verder was dit moontlik om "n drankie te vervaardig met die korrels wat baie dieselfde is as tradisionele Kepi.

Thesis (MSc Food Sc)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: The genus Cronobacter (Enterobacter sakazakii) contains opportunistic pathogens that can cause a severe form of neonatal meningitis, necrotising enterocolitis and septicaemia. Cronobacter infections have been reported in all age groups, however, immunocompromised infants are more susceptible to these infections. Furthermore, Cronobacter strains have been reported to show differences in sensitivity to antibiotics and virulence. These differences led to the reclassification of Cronobacter and currently the genus contains five distinct species, namely Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter turicensis, Cronobacter dublinensis and Cronobacter muytjensii. As this reclassification was only accepted recently, there are not many typing methods optimised for differentiation between the five Cronobacter species. Typing of Cronobacter strains are important as the species may be diverse regarding their virulence. Cronobacter strains have been isolated from infant formula milk (IFM), the environment of an IFM processing facility and fresh produce in South Africa. However, little is known about the phylogeny and prevalence of these strains. The aim of this study was to classify 24 South African Cronobacter strains (previously identified as E. sakazakii) and to evaluate the phylogeny of the isolates based on the 16S ribosomal RNA (rRNA) and rpoA genes. All 24 South African strains were identified as Cr. sakazakii despite a wide variety of isolation sources. Other studies have also found that irrespective of the isolation source, the majority of Cronobacter strains are identified as Cr. sakazakii. The South African strains were found to be phylogenetically closely related. However, two distinct clusters separated at a 93 % confidence level were observed in the Cr. sakazakii group based on the 16S rRNA gene analysis. Strains of Cr. sakazakii, Cr. dublinensis, Cr. turicensis and Cr. muytjensii were differentiated from each other with sequence data of the 16S rRNA and rpoA genes, but it was not possible to differentiate between Cr. sakazakii and Cr. malonaticus. The phylogram based on the rpoA gene sequences did separate Cr. malonaticus and Cr. sakazakii strains, however, the clusters were separated with a low bootstrap value of 70 %. Phylogenetic analysis based on the rpoA and 16S rRNA genes were, therefore, not sufficient to distinguish between all the Cronobacter species. The sequence data of these two genes can be used to differentiate between the Cronobacter strains when used in combination with malonate utilisation analysis. A PCR-RFLP method was subsequently developed to facilitate the simultaneous differentiation between all five Cronobacter species. The PCR-RFLP assay was based on the amplification of the rpoB gene followed by the combined digestion with restriction endonucleases Csp6I and HinP1I. Unique profiles for each of the five Cronobacter species were obtained and it was also possible to differentiate between Enterobacteriaceae and Cronobacter strains. Furthermore, two strains which were identified as Cr. sakazakii with sequencing based on the 16S rRNA and rpoA genes had PCR-RFLP profiles identical to that of Cr. malonaticus. Sequencing based on the rpoB gene and additional biochemical analysis with malonate broth confirmed the identities of these two strains as Cr. malonaticus. This PCR-RFLP assay is, therefore, an accurate typing method that ensures rapid differentiation between the five species of Cronobacter. / AFRIKAANSE OPSOMMING: Die Cronobacter genus (Enterobacter sakazakii) bevat opportunistiese patogene wat 'n ernstige vorm van neonatale meningitis, enterokolitis en septisemie kan veroorsaak. Cronobacter infeksies is al in alle ouderdomsgroepe aangemeld, maar immuungekompromitteerde babas is die meeste vatbaar vir hierdie infeksies. Verder toon Cronobacter spesies verskille in virulensie en sensitiwiteit vir antibiotika. Hierdie verskille het gelei tot die herklassifikasie van Cronobacter en tans bestaan die genus uit vyf afsonderlike spesies, naamlik Cronobacter sakazakii, Cronobacter malonaticus, Cronobacter turicensis, Cronobacter dublinensis en Cronobacter muytjensii. Aangesien hierdie herklassifikasie slegs onlangs aanvaar is, is daar nie baie metodes wat geskik is vir onderskeiding tussen die vyf Cronobacter spesies nie. Onderskeiding tussen Cronobacter spesies is belangrik omdat die spesies verskillend kan wees met betrekking tot hulle virulensie. Cronobacter is geisoleer uit baba formule melk (BFM), die omgewing van 'n BFM fabriek en vars produkte in Suid-Afrika. Daar is egter nie baie bekend oor die filogenie en voorkoms van hierdie isolate nie. Die doel van hierdie studie was om 24 Suid-Afrikaanse Cronobacter stamme (voorheen geïdentifiseer as E. sakazakii) te klassifiseer en die filogenie van die isolate te evalueer gebaseer op die 16S ribosomale RNS (rRNS) en rpoA gene. Al 24 Suid-Afrikaanse stamme is geïdentifiseer as Cr. sakazakii ten spyte van 'n wye verskeidenheid isolasie bronne. Ander studies het ook gevind dat, ongeag die isolasie bron, die meerderheid van Cronobacter stamme as Cr. sakazakii geïdentifiseer word. In hierdie studie is gevind dat die Suid-Afrikaanse stamme filogeneties nou verwant is. Op grond van die 16S rRNA geen analise is die Cr. sakazakii stamme egter in twee afsonderlike groepe gedeel met 'n 93% vertrouens vlak. Dit was moontlik om stamme van Cr. sakazakii, Cr. dublinensis, Cr. turicensis en Cr. muytjensii van mekaar te onderskei met die DNS volgorde data van die 16S rRNA en rpoA gene, maar geen onderskeid tussen Cr. sakazakii en Cr. malonaticus stamme was moontlik nie. Die filogram gebaseer op die rpoA DNS volgorde data het aparte takke vir Cr. malonaticus en Cr. sakazakii stamme getoon, maar die twee takke is met ‘n lae vertrouens waarde van slegs 70 % geskei. Filogenetiese analise gebaseer op die rpoA en 16S rRNA gene is dus nie voldoende om te onderskei tussen al die Cronobacter spesies nie. Die DNS volgorde data van hierdie twee gene sou egter gebruik kon word om te onderskei tussen die Cronobacter spesies wanneer dit gebruik word in kombinasie met malonaatbenutting-analises. 'n Polimerase ketting reaksie (PKR) beperkings fragment lengte polimorfisme (BFLP) metode is ontwikkel om die gelyktydige onderskeiding tussen al vyf Cronobacter spesies te fasiliteer. Die PKR-BFLP metode is gebaseer op die vermeerdering van die rpoB geen gevolg deur die gesamentlike vertering met die beperkingsensieme, Csp6I en HinP1I. Unieke profiele vir elk van die vyf Cronobacter spesies is verkry en dit was ook moontlik om tussen Enterobacteriaceae en Cronobacter spesies te onderskei. Verder het twee stamme wat as Cr. sakazakii geïdentifiseer is met DNS volgordebepaling van die 16S rRNA en rpoA gene, PKR-BFLP profiele identies aan dié van Cr. malonaticus getoon. DNS volgordebepaling van die rpoB geen en ‘n addisionele biochemiese toets met malonaat sop het die identiteit van hierdie twee stamme as Cr. malonaticus bevestig. Hierdie PKR-BFLP is dus 'n akkurate metode wat vinnige onderskeid tussen die vyf spesies van Cronobacter kan verseker.

Thesis (MSc Food Sc)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: The use of near infrared (NIR) hyperspectral imaging for distinguishing between pre-germinated and non pre-germinated barley, wheat and sorghum kernels and, the effect of kernel shape on hyperspectral images, have been investigated. Two sample sets were imaged. The first sample set was divided into six subsets; these subsets were treated with water and left to pre-germinate for different times (0, 6, 9, 12, 18 and 24 hrs). Subset viability was determined with the tetrazolium test. The second sample set was divided into seven subsets, treated with water and left to pre-germinate for 0, 3, 6, 9, 12, 18, 24 or 30 hrs. Individual kernel viability was determined with the tetrazolium test. NIR hyperspectral images were acquired using two different SisuCHEMA hyperspectral imaging systems. The first system acquired images with a 150 9m spatial resolution (first sample set) and the second system acquired images with a 30 9m spatial resolution (second sample set). Principal component analysis (PCA) was performed and a distinction between pre-germinated and non pre-germinated kernels was illustrated in PCA score images. Loading line plots showed that the main compounds contributing to spectral variation were starch, water and protein. These compounds were related to starch and protein hydrolysis. The distinction between pre-germinated and non pre-germinated kernels observed in the 30 9m spatial resolution images indicated NIR hyperspectral imaging was perhaps sensing incomplete endosperm degradation. Some kernels determined as pre-germinated by the tetrazolium test had the same chemical composition according to the score image as non pre-germinated kernels in the 30 9m spatial resolution images. A partial least squares discriminant analysis (PLS-DA) model with two classes (pre- germinated and non pre-germinated) was developed for each of the cultivars of the first sample set. The two classes were assigned in principal component (PC) 1 vs. PC 5 score plots. The model created for the barley cultivars resulted in excessive false positives and false negatives. The prediction results of wheat cultivars revealed that the model had a classification rate of 81% for the non pre-germinated class and 93% for the pre-germinated class. The sorghum prediction results revealed that the model correctly predicted 97% of the non pre-germinated class and 93% of the pre-germinated class. Two different PLS-DA models were developed for one image of each cultivar of the 30 9m spatial resolution images. The first model was developed by assigning each kernel in the score image and the second model was developed by assigning pixels in the score plot to either the pre- germinated or non pre-germinated class. Model 1 resulted in excessive false negatives. Model 2 resulted in excessive false positives. The differences between pre-germinated and non pre-germinated kernels were only observed in higher (PC 5 and 6) order PCs of the 150 9m spatial resolution images. The lower (PCs 1 to 4) order PCs (of each commodity) were subsequently examined with the aid of classification gradients. Kernel shape effects were observed in these PCs. The use of NIR hyperspectral imaging for distinguishing between pre-germinated and non pre-germinated grain kernels shows promise. / AFRIKAANSE OPSOMMING: Die gebruik van naby infrarooi (NIR) hiperspektrale beeld-analise is geëvalueer om onderskeid te tref tussen voor-ontkiemde en nie-voor-ontkiemde gars, koring en sorghum korrels. Die effek van korrelvorm op hiperspektrale beelde is ook geëvalueer. Die eerste stel graan-monsters is gebruik vir 150 9m ruimtelike resolusie beelde en die tweede stel is gebruik vir 30 9m ruimtelike resolusie beelde. Die eerste kultivar stel is verdeel in ses sub-stelle en met gedistilleerde water behandel vir 0, 6, 9, 12, 18 en 24 hr. Sub-stel lewensvatbaarheid is met die tetrazolium toets vasgestel. Elke kultivar in die tweede stel is in sewe sub-stelle verdeel en is vir 0, 3, 6, 9, 12, 18, 24 of 30 hr geïnkubeer. Individuele korrel lewensvatbaarheid is met die tetrazolium toets vasgestel. NIR hiperspektrale beelde is verkry deur gebruik te maak van twee verskillende SisuCHEMA kameras. Die verskillende kameras is gebruik om verskillende resolusie (30 en 150 9m ruimtelike resolusie) beelde te verkry. Hoofkomponent analise (HKA) is uitgevoer en ’n verskil tussen voor- ontkiemde en nie-voor-ontkiemde korrels is waargeneem in die 150 9m ruimtelike resolusie beelde. HK ladings stippe het water, stysel en proteïene uitgesonder as die verbindings wat bydrae het tot spektrale variasie. ’n Verskil tussen die voor-ontkiemde korrels en nie-voor-ontkiemde korrels is ook gesien vir die 30 9m ruimtelike resolusie beelde. Dit is egter ook waargeneem dat sommige korrels as voor-ontkiem bepaal is deur die tetrazolium toets, maar dié korrels het dieselfde chemiese samestelling volgens die punte beeld as nie-voor-ontkiemde korrels. Onvolledige endosperm hidrolise is ’n moontlike verduideliking vir die verskynsel. Die verbindings wat bygedra het tot die variasie is water, stysel en proteïene. ’n Parsiële kleinste kwadrate diskriminant analise (PKW-DA) model met twee klasse is ontwikkel vir elke kultivar van die 150 9m ruimtelike resolusie beelde. Die klasse is aangewys in the punte stip. Die model met die hoogste variasie in Y is gekies om die ander kultivars van dieselfde kommoditeit te voorspel. The PKW-DA resultate vir die gars kultivars het getoon dat die model vals positiewes en vals negatiewes opgelewer het. Die koring PKW-DA model het ’n klassifikasie koers van 81% vir die nie-voor-ontkiemde klasse en 93% vir die voor-ontkiemde klasse opgelewer. The PKW-DA resultate vir sorghum het getoon dat die model ’n klassifikasie koers van 97% vir die nie-voor-ontkiemde klasse en 93% vir die voor-ontkiemde klasse opgelewer. Twee verskillende PKW-DA modelle is ontwikkel vir elke beeld van elke kultivar van die 30 9m ruimtelike resolusie beelde. Die eerste model is ontwikkel deur elke korrel in die punte beeld aan te wys tot een van twee klasse en die tweede model is ontwikkel deur die beeldelemente in die punte stip tot een van twee klasse toe te skryf. Model 1 het vals negatiewes opgelewer en model 2 vals positiewes. Die verskille tussen die nie-voor-ontkiemde en voor-ontkiemde korrels is eers verduidelik in hoër orde HK van die 150 9m ruimtelike resolusie beelde. Die laer orde HK is dus ondersoek vir hul bydrae tot spektrale variasie met die hulp van klassifikasie gradiënte. Korrel vorm effekte is waargeneem. Die gebruik van NIR hiperspektrale beelding om onderskeid te tref tussen voor-ontkiemde en nie-voor-ontkiemde graan korrels, lyk belowend.

Thesis (MSc Food Sc)--University of Stellenbosch, 2011. / ENGLISH ABSTRACT: Near infrared hyperspectral imaging (NIR HSI) in conjunction with multivariate image analysis was evaluated for the detection of millet and buckwheat flour in ground black pepper. Additionally, midinfrared (MIR) spectroscopy was used for the quantification of millet and buckwheat flour in ground black pepper. These techniques were applied as they allow non-destructive, invasive and rapid analysis. Black pepper and adulterant (either millet or buckwheat flour) mixtures were made in 5% (w/w) increments spanning the range 0-100% (w/w). The mixtures were transferred to eppendorf tube holders and imaged with a sisuChema short wave infrared (SWIR) pushbroom imaging system across the spectral range of 1000–2498 nm. Principal component analysis (PCA) was applied to pseudo-absorbance images for the removal of unwanted data (e.g. background, shading effects and bad pixels). PCA was subsequently applied to the ‘cleaned’ data. An adulterant concentration related gradient was observed in principal component one (PC1) and a difference between black pepper adulterated with buckwheat and millet was noted in PC4. Four absorption peaks (1461, 2241, 2303 and 2347 nm) were identified in the loading line plot of PC1 that are associated with protein and oil. The loading line plot of PC4 revealed absorption peaks at 1955, 1999, 2136 and 2303 nm, that are related to protein and oil. Partial least squares discriminant analysis (PLS-DA) was applied to NIR HSI images for discrimination between black pepper adulterated with varying amounts of adulterant (millet or buckwheat). The model created with millet adulterated black pepper samples had a classification accuracy of 77%; a classification accuracy of 70% was obtained for the buckwheat adulterated black pepper samples. An average spectrum was calculated for each sample in the NIR HSI images and the resultant spectra were used for the quantification of adulterant (millet or buckwheat) in ground black pepper. All samples were also analysed using an attenuated total reflectance (ATR) Fourier transform (FT) – infrared (IR) instrument and MIR spectra were collected between 576 and 3999 cm-1. PLS regression was employed. NIR based predictions (r2 = 0.99, RMSEP = 3.02% (w/w), PLS factor = 4) were more accurate than MIR based predictions (r2 = 0.56, RMSEP = 19.94% (w/w), PLS factors = 7). Preprocessed NIR spectra revealed adulterant specific absorption bands (1743, 2112 and 2167 nm) whereas preprocessed MIR spectra revealed a buckwheat specific signal at 1574 cm-1. NIR HSI has great promise for both the qualitative and quantitative analysis of powdered food products. Our study signals the beginning of incorporating hyperspectral imaging in the analysis of powdered food substances and results can be improved with advances in instrumental development and better sample preparation. / AFRIKAANSE OPSOMMING: Die gebruik van naby infrarooi hiperspektrale beelding (NIR HB) tesame met veelvoudige beeldanalise is ondersoek vir die opsporing van stysel-verwante produkte (giers en bokwiet) in gemaalde swart pepper. Middel-infrarooi (MIR) spektroskopie is addisioneel gebruik vir die kwantifisering van hierdie stysel-verwante produkte in swart pepper. Albei hierdie tegnieke is toegepas aangesien dit deurdringend van aard is en dit bied nie-destruktiewe sowel as spoedige analise. Swart pepper en vervalsingsmiddel (giers of bokwiet) mengsels is uitgevoer in 5% (m/m) inkremente tussen 0 en 100% (m/m). Eppendorfbuishouers is met die mengsels gevul en hiperspektrale beelde is verkry deur die gebruik van ‘n sisuChema SWIR (kortgolf infrarooi) kamera met ‘n spektrale reikwydte van 1000–2498 nm. Hoofkomponent-analise (HK) is toegepas op pseudo-absorbansie beelde vir die verwydering van ongewenste data (bv. agtergrond, skadu en dooie piksels). Hoofkomponent-analise is vervolgens toegepas op die ‘skoon’ data. Hoofkomponent (HK) een (HK1) het die aanwesigheid van ‘n vervalsingsmiddel konsentrasie verwante gradient getoon terwyl HK4 ‘n verskil getoon het tussen swart pepper vervals met giers en bokwiet. Vier absorpsiepieke (1461, 2241, 2303 en 2347 nm) was geïdentifiseer binne die HK lading stip van HK1 wat met proteïen en olie geassosieer kon word. Die HK lading stip van HK4 het absorpsipieke by 1955, 1999, 2136 en 2303 nm aangedui wat verband hou met proteïen en olie. Parsiële kleinste waarde diskriminant-analise (PKW-DA) is toegepas op die hiperspektrale beelde vir die moontlike onderskeiding tussen swart pepper vervals met verskeie hoeveelhede vervalsingsmiddel (giers of bokwiet). ‘n Klassifikasie koers van 77% is verkry vir die model ontwikkel met giers vervalsde swart pepper terwyl die model ontwikkel met bokwiet vervalsde swarte pepper ‘n klassifikasie koers van 70% bereik het. ‘n Gemiddelde spektrum is bereken vir elke monster in die hiperspektrale beelde en die resulterende spektra is gebruik vir die kwantifisering van vervalsingsmiddels (giers of bokwiet) in gemaalde swart pepper. ‘n ATR FT-IR instrument met spektrale reikwydte van 576-3999 cm-1 is additioneel gebruik vir die analise van alle monsters. Parsiële kleinste waarde regressie is gebruik vir kwantifikasie doeleindes. NIR gebasseerde voorspellings (r2 = 0.99, RMSEP = 3.02% (m/m), PLS faktore = 4) was meer akkuraat as die MIR gebasseerde voorspellings (r2 = 0.56, RMSEP = 19.94% (m/m), PLS faktore = 7). Vooraf behandelde NIR spektra het vervalsingsmiddel verwante absorpsiepieke (1743, 2112 en 2167 nm) aangetoon terwyl vooraf behandelde MIR spektra ‘n bokwiet verwante absorpsiepiek by 1574 cm-1 aangedui het. NIR HB toon goeie potensiaal vir beide kwalitatiewe en kwantitatiewe analise van gepoeierde voedsel produkte. Ons studie kan gesien word as die begin van die inkorporasie van hiperspektrale beelding in die analise van gepoeierde voedsel material en verbeterde resulte kan verkry word deur die vordering in instrumentasie ontwikkeling en verbeterde monstervoorbereiding.