Thesis (MScFoodSc)--Stellenbosch University, 2004. / ENGLISH ABSTRACT: The regular consumption of probiotics is becoming a recognized trend in the food industry due to several reported health benefits. A probiotic is defined as a live microbial feed supplement that beneficially affects the host animal by improving its intestinal microbial balance. A wide variety of probiotic food products are available on the South African market and comprise an assortment of fermented milks, as well as lyophilized preparations in tablet or capsule form. Strains of Lactobacillus acidophilus and Bifidobacterium species are mostly used as probiotic microbes in the industry due to their health enhancing effect. The survival of sensitive probiotic microbial species in food matrices are influenced by various factors such as oxygen concentration, pH levels and manufacturing and storage conditions. These should be considered and monitored as the South African food and health regulations stipulate that probiotic microbes should be present at a concentration of 10⁶ cfu.ml ̄ ¹' in order to exert a beneficial effect. Some health benefits are also correlated to specific microbial species and strains and these factors have resulted in the need for the rapid and accurate identification of probiotic microbes present in food products. The probiotic microbes present in probiotic yoghurts and supplements have in the past been identified using traditional methods such as growth on selective media, morphological, physiological and biochemical characteristics. However, even some of the most sophisticated cultural-dependant techniques are not always sufficient for the identification and classification of especially Bifidobacterium, as well as closely related Lactobacillus species. Molecular techniques are more often employed for the rapid and accurate detection, identification and characterization of microbial species present in food products. The aim of this study was to detect and identify the probiotic species present in various commercial South African yoghurts and lyophilized preparations using peR-based DGGE analysis. A 200 bp fragment of the V2-V3 region of the 16S rRNA gene was amplified and the peR fragments were resolved by DGGE. The unique fingerprints obtained for each product were compared to two reference markers A and B in order to identify the bands present. The results obtained were verified by species-specific peR, as well as sequence analyses of bands that could not be identified when compared to the reference markers. Only 54.5% of the South African probiotic yoghurts that were tested did contain all the microbial species as were mentioned on the labels of these products, compared to merely one third (33.3%) of the lyophilized probiotic food supplements. Some Bifidobacterium species were incorrectly identified according to some product labels, while other products contained various microbes that were not mentioned on the label. Sequence analysis confirmed the presence of a potential pathogenic Streptococcus species in one of the yoghurt products and in some instances the probiotic species claimed on the labels were non-scientific and misleading. The data obtained in this study showed that the various South African probiotic products tested were of poor quality and did not conform to the South African regulations. peR-based DGGE analysis proofed to be a valuable approach for the rapid and accurate detection and identification of the microbial species present in South African probiotic products. This could help with future implementation of quality control procedures in order to ensure a reliable and safe probiotic product to the consumer. / AFRIKAANSE OPSOMMING: Die gereelde inname van probiotiese produkte is besig om In erkende tendens in die voedselindustrie te word, as gevolg van verskeie gesondheidsvoordele wat daaraan gekoppel word. In Probiotika word gedefinieer as In voedingsaanvulling wat uit lewendige mikrobes bestaan en wat In voordelige effek op mens of dier het deur In optimale mikrobiese balans in die ingewande te handhaaf. In Wye verskeidenheid probiotiese voedselprodukte is tans beskikbaar op die Suid- Afrikaanse mark. Hierdie bestaan hoofsaaklik uit verskeie gefermenteerde melkprodukte asook 'n reeks tablette en kapsules wat probiotiese mikrobes in gevriesdroogde vorm bevat. Lactobacillus acidophilus tipes en Bifidobacterium spesies word die algemeenste in die voedselindustrie gebruik aangesien hierdie spesifieke mikrobes bekend is om goeie gesondheid te bevorder. Die oorlewing van sensitiewe probiotiese mikrobiese spesies in voedsel matrikse word beïnvloed deur faktore soos suurstof konsentrasie, pH-vlakke en vervaardigings- en opbergings kondisies. Hierdie faktore moet in aanmerking geneem word en verkieslik gemonitor word aangesien die Suid-Afrikaanse voedsel en gesondheids regulasies stipuleer dat probiotiese mikrobes teen In konsentrasie van 10⁶ kolonie vormende eenhede per ml teenwoordig moet wees om In voordelige effek te toon. Sommige gesondheidsvoordele word direk gekoppel aan spesifieke mikrobiese spesies en spesie-tipes. Hierdie faktore het gelei tot In groot aanvraag na vinnige en akkurate metodes vir die identifikasie van probioties mikrobes in voedselprodukte. Die probiotiese mikrobes teenwoordig in probiotiese joghurts en ook die gevriesdroogde vorms in tablette en kapsules, was al geïdentifiseer deur gebruik te maak van tradisionele metodes soos groei op selektiewe media, morfologiese, fisiologiese en biochemiese eienskappe. Selfs van die mees gesofistikeerde kultuur-afhanklike tegnieke is egter nie altyd voldoende vir die identifikasie en klassifikasie van veral Bifidobacterium en na-verwante Lactobacillus spesies nie. Molekulêre metodes word dikwels aangewend vir die vinnige en akkurate deteksie, identifikasie en karakterisering van mikrobes teenwoordig in voedselprodukte. Die doel van hierdie studie was om die probiotiese mikrobes teenwoordig in verskeie Suid-Afrikaanse joghurts en gevriesdroogde aanvullings, te identifiseer deur gebruik te maak van polimerase kettingreaksie (PKR)-gebaseerde denaturerende gradiënt jelelektroforese (DGGE) analise. 'n PKR fragment van 200 bp van die V2-V3 gedeelte van die 16S ribosomale RNS (rRNS) geen is geamplifiseer, en die PKR fragmente is geskei met behulp van DGGE. Die unieke vingerafdrukke wat verkry is vir elke produk is teen twee verwysings merkers A en B vegelyk om die bande teenwoordig in die profiele te identifiseer. Die resultate is bevestig deur spesies-spesifieke PKR en ook deur die ketting volgordes van die DNS fragmente te bepaal wat nie geïdentifiseer kon word deur vergelyking met die verwysings merkers nie. Slegs 54.5% van die Suid-Afrikaanse probiotiese joghurts wat getoets is het al die mikrobiese spesies bevat soos aangedui was op die etikette van hierdie produkte, teenoor slegs 'n derde (33.3%) van die gevriesdroogde voedingsaanvullings. Sekere Bifidobacterium spesies is verkeerd geïdentifiseer op sommige van die produk etikette, terwyl ander produkte verskeie mikrobes bevat het wat nie op die etiket aangedui was nie. 'n Potensiële patogeniese Streptococcus spesie is in een van die joghurt produkte gevind soos bevestig deur DNS kettingvolgorde bepalings. In sommige gevalle was die probiotiese spesienaam wat aangedui is op die etiket onwetenskaplik en misleidend. Die resultate wat uit hierdie studie verkry is dui aan dat die Suid-Afrikaanse probiotiese produkte wat getoets is van 'n swak gehalte is en nie aan die Suid- Afrikaanse regulasies voldoen nie. Daar is getoon dat PKR-gebaseerde DGGE analise 'n waardevolle tegniek kan wees vir die akkurate deteksie en identifisering van die mikrobiese spesies teenwoordig in probiotiese produkte. Dit kan help met die toekomstige implementering van kwaliteitskontrolerings prosedures om 'n mikrobiologiese betroubare en veilige produk aan die verbruiker te verseker.

Thesis (MSc Food Sc )--Stellenbosch University, 2000. / ENGLISH ABSTRACT: This study shows Fourier transform near infrared (FT-NIR) spectroscopy's application on wine, fruit and dried fruit for quantitative determinations or as a discriminative method for classification purposes. During wine production optimum yeast growth, resulting in healthy alcohol fermentation rates, is monitored by the amount of free amino nitrogen (FAN) present in the must. The status of malolactic fermentation (MLF) in Chardonnay wines is monitored by determining the degree of conversion of malic to lactic acid. Ethyl carbamate (EC), a suspected carcinogen, is mainly formed during ageing of wine and is restricted by legislation in some countries. It is therefore necessary to determine the EC content in wine. Fourier transform near infrared spectroscopy can be used on wine as a rapid method to measure the °Brix (residual sugars) content of must and to discriminate between different must samples in terms of their FAN values. It can also be used as a rapid method to discriminate between Chardonnay wine samples in terms of the MLF status and between table wine samples in terms of the EC content. Calibrations were derived and it was found that a very strong correlation existed in the sample set for the FT-NIR spectroscopic predictions for °Brix (r = 0.99, SEP = 0.31%), but poorer correlations for the FAN (r = 0.405, SEP = 275%), malic acid (r = 0.64, SEP = 1.02%), lactic acid (r = 0.61, SEP = 1.35%) and EC predictions (r = 0.47, SEP = 3.6%). When soft independent modelling by class analogy (SIMCA) was applied as a discriminative method, the must and wine samples were classified in terms of their FAN and EC values and MLF status, respectively, obtaining results with recognition rates exceeding 80%. The canning of peaches has become a significant industry in South Africa, producing approximately 4.1 million cartons per year. Fourier transform near infrared spectroscopy was applied as an alternative non-destructive method for the quantitative determination of the total soluble solid (TSS) content of whole fresh peaches. The TSS content of fresh clingstone peaches is an indication of the internal quality, maturity and perceived sweetness of the fruit for the peach canning industry. By determining the TSS, fresh peaches can be graded and the farmers compensated accordingly. Results obtained by building QUANT+™calibrations for the TSS content (r = 0.96, SEP = 0.55%) showed acceptable accuracy and can replace the present destructive methods. Peak periods during the harvesting season necessitate storage of peaches for up to three weeks before canning. Approximately 5 - 10% of the peaches stored, disintegrate during canning due to loose skin, large stone cavities, soft flesh and rot. The storage potential of fresh clingstone peaches can be successfully predicted with FT-NIR and SIMCA models, using subjective internal quality evaluations. Results with recognition rates exceeding 80% were obtained in most cases and this method proved useful as a non-destructive method of quality assessment. By applying this method, losses caused when storing peaches with poor storage quality will be reduced. The golden sultana industry plays an important role in the dried fruit exporting market of South Africa. Due to the large numbers of consignments that must be checked upon arrival, and the need for rapid decision making during processing, it is essential to replace the present time-consuming analytical methods. Fourier transform near infrared spectroscopy was used as a rapid, analytical technique to determine whether the S0₂ and moisture contents of sultanas are within specifications upon arrival at the factory and during processing. High positive correlation was found between the measured values and those predicted by FT-NIR spectroscopy for S0₂ (r = 0.99, SEP = 24.09%) and moisture (r = 0.99, SEP = 0.051 %) contents. / AFRIKAANSE OPSOMMING: Hierdie studie dui op Fourier transformasie naby-infrarooi (FT-NIR) spektroskopie se toepassing op wyn, vrugte en droëvrugte vir die uitvoer van kwantitatiewe bepalings of vir klassifikasie doeleindes om as 'n diskriminasie metode te dien. Gedurende wynproduksie word die optimum groei van giste wat lei tot 'n gesonde alkohol fermentasie gemonitor deur die hoeveelheid vry-aminostikstof (VAS) wat in die mos teenwoordig is te bepaal. Die status van appelmelksuurgisting (AMG) in Chardonnay wyne word gemonitor deur die mate van omskakeling van appelsuur na melksuur te bepaal. Etielkarbamaat (EK), 'n vermoede karsinogeen wat hoofsaaklik in verouderde wyne voorkom, word in sekere lande deur wetgewing beperk en dus die bepaling van die EK inhoud van wyne noodsaak. Fourier transformasie naby-infrarooi spektroskopie kan op mos toegepas word as 'n vinnige metode vir die bepaling van °Brix (residuele suiker) en om tussen die monsters te onderskei in terme van hulle VAS inhoud. FT-NIR kan ook gebuik word as 'n vinnige metode om tussen Chardonnay monsters te onderskei op grond van die status van AMG en tussen tafelwyn monsters op grond van die EK inhoud. Kalibrasies is ontwikkel en daar is gevind dat baie sterk korrelasies bestaan in die monsterstel vir die FT-NIR spektroskopiese voorspelling van °Brix (r = 0.99, SEP = 0.31%), maar swakker korrelasies vir die VAS (r = 0.405, SEP = 275%), appelsuur (r = 0.64, SEP = 1.02%), melksuur (r = 0.61, SEP = 1.35%) en EK voorspellings (r = 0.47, SEP = 3.6%). Met die toepassing van soft independent modelling by class analogy (SIMCA) as diskriminasie metode, is die mos- en wynmonsters geklassifiseer op grond van hul VAS en EK waardes en die status van AMG, en herkenningswaardes van bo 80% is onderskeidelik behaal. Die inmaak van perskes het 'n beduidende industrie in Suid-Afrika geword en produseer jaarliks ongeveer 4.1 miljoen kartonne. Fourier transformasie nabyinfrarooi spektroskopie is toegepas as alternatiewe, nie-beskadigende metode om kwantitatiewe bepalings van die totale oplosbare vastestowwe (TOV) inhoud van heel vars perskes, te doen. Vir die perske inmaak-industrie is die TOV inhoud van vars taaipitperskes 'n aanduiding van interne kwaliteit, rypheid en die soetheid van die vrugte. Vars perskes kan gradeer word deur die TOS te bepaal en sodoende kan boere oreenkomstige vergoeding ontvang. Resultate' wat verkry is deur QUANT+™ kalibrasies vir TOS inhoud te ontwikkel (r = 0.96, SEP = 0.55%), dui op aanvaarbare akkuraatheid en kan die huidige metodes vervang. Tydens oestyd kom piektye voor wanneer dit soms nodig is om perskes vir tot drie weke op te berg voordat dit ingemaak kan word. Ongeveer 5 tot 15% van hierdie opgebergte perskes disintegreer tydens inmaak omdat opberging lei tot defekte in die perskes soos skille wat loskom, groot pitholtes, sagte vleis en vrot. Die opbergingspotentiaal van vars taaipitperskes kan suksesvol voorspel word deur FTNIR en SIMCA modelle te bou en subjektiewe interne kwaliteitsevaluerings daarop toe te pas. Herkenningsresultate wat 80% in die meeste gevalle oorskry, is behaal wat hierdie metode as 'n suksesvolle nie-beskadigende kwaliteitbepalingsmetode bewys. Hierdie metode sal verliese kan beperk wat voorkom as gevolg van opberging van perskes met swak opbergingskwaliteit. Die goue sultana industrie speel 'n belangrike rol in die droë vrugte uitvoermark van Suid-Afrika. As gevolg van die hoeveelheid monsters wat gelyktydig getoets moet word en besluite wat vinnig geneem moet word tydens prosessering, is dit belangrik om die huidige tydrowende analitiese metodes te vervang. Fourier transformasie naby-infrarooi spektroskopie is gebruik as 'n vinnige, analitiese tegniek om tydens ontvangs by die fabriek en gedurende prosessering te bepaal of die S0₂- en voginhoud van goue sultanas binne die spesifikasies val. Goeie positiewe korrelasie is gevind tussen die bepaalde en voorspelde FT-NIR spektroskopiese waardes vir SO₂- (r = 0.99, SEP = 24.09%) en voginhoud (r = 0.99, SEP = 0.051 %).