Thesis (MSc Food Sc )--Stellenbosch University, 2000. / ENGLISH ABSTRACT: Fermented fish products are popular food products mainly consumed and produced in Southeast Asia. These products are not produced in South Africa, and those available to the public are imported. The main action during the production and fermentation of this sort of product, is that of proteolysis, either by the bacteria or enzymes naturally present in the fish. The prevalent microbes present in six fermented fish samples from Bangkok (Thailand) and seven from Khon Kaen (Thailand), were determined, and using numerical methods, clustered into similarity groups using the calculated dendrogram .distance (Do) technique to determine their relation to reference strains. Forty-seven different bacterial strains were isolated, but no yeasts, moulds or lactic acid bacteria were found. Five Gram-negative, oxidase-positive species, five different Staphylococcus species and nine different endospore-forming species of the genus Bacillus, were isolated and identified using the API systems. The data indicated that members of the genus Bacillus were the prevalent organisms in all the products examined. The isolates were also scanned for general enzyme activity using the API Zym technology, and the production of proteases was investigated using the Standard Methods Caseinate and the Universal Protease Substrate methods. It was found that most of the isolated organisms produced protease, which is of major importance in the production of fermented fish products. Proteolytic cultures from the fermented fish products, as well as lactic acid starters, were used in the production of a fermented fresh water fish product. Production parameters including: glucose, inoculum, moisture content and incubation time, were evaluated in order to select optimum fermentation conditions. Fermentation efficiency was determined by measuring the final pH, titratable acid and the free amino nitrogen content. Optimum efficiency was obtained with 5% (w/w) added glucose at a moisture level of 150 ml water per 100 g fish. A factorial design (3 x 3 x 3) was used to indicate viable trends to facilitate optimisation of the fermentation process. The main effects, two-factor and three-factor interactions were calculated. Optimum trends obtained were a glucose concentration of 5% (wlw) , inoculum concentration of 1x10⁸ kve.ml ̄ ¹, an incubation period of 15 days and temperature of 30°C. Three lactic acid starters (226 - Lactobacillus plantarum, 140 - Lactococcus diacetylactis and 407 - Pediococcus cerevisiae) were selected as they produced some of the best fermentation results and are safe to use in food. It was found that a combination of all three strains (226, 140 and 407) yielded the best results. By using the above parameters, an acceptable product was produced in terms of consistency, colour and aroma. Further studies need to be conducted to optimise the safety of the product as well as the flavour, both chemically and sensorically optimisation of the product. / AFRIKAANSE OPSOMMING: Gefermenteerde visprodukte is populere voedselprodukte in die lande van Suidoos-Asie. Die produkte word nie in Suid-Afrika geproduseer nie, maar slegs ingevoer. Die hoof aksie tydens die fermentasie proses is proteolise deur die bakteriee en ensieme wat natuurlik teenwoordig is in vis. Die oorheersende mikrobes teenwoordig in ses gefermenteerde vis produkte van Bangkok (Thailand) en sewe van Khon Kaen (Thailand), is bepaal. Numeriese metodes is gevolg om die isolate in groepe te sorteer en te groepeer deur gebruik te maak van die berekende dendrogram afstand (Do) tegniek om hul verwantskap ten opsigte van die verwysingsorganisme te bepaal. Sewe-en-veertig verskillende bakteriee is ge·isoleer, maar geen fungi of melksuurbakteriee is ge·identifiseer nie. Vyf Gram-negatiewe, oksidase-positiewe spesies, vyf verskillende Staphylococcus spesies en nege verskillende endospoorvormende spesies van die genus Bacillus, is geisoleer en ge·identifiseer deur gebruik te maak van die API CHB sisteme. Die data het getoon dat lede van die genus Bacillus die oorheersende organismes was. Die isolate is daarna ondersoek vir algehele ensiemaktiwiteit deur van die API Zym tegnologie gebruik te maak. Daar is veral klem gelê op die protease aktiwiteit en dit is gemeet deur van die "Standard Methods Caseinate Agar" metode asook die "Universal Protease Substrate" metodes gebruik te maak. Daar is gevind dat die oorgrote meerderheid organismes proteolitiese ensieme produseer wat belangrik is in die produksie van gefermenteerde visprodukte. Kulture wat ge·isoleer is uit gefermenteerde visprodukte asook melkssuurkulture is gebruik vir die produksie van 'n gefermenteerde varswater visproduk. Produksieparameters insluitende: glukose-, inokulum- en voginhoud asook inkubasie tyd is ondersoek om die optimum fermentasie kondisies te bepaal. Optimum effektiwiteit is gevind by 'n 5% glukose konsentrasie en vogvlakke van 150 ml water per 100 9 vis. 'n Faktoriale ontwerp (3 x 3 x 3) is gebruik om die optimum kondisies te bepaal. Die hoof effekte asook die twee faktor en drie faktor interaksies is bereken. Optimum neigings is gevind by 'n glukose konsentrasie van 5%, inokulum konsentrasie van 1x10⁸ kve.ml ̄ ¹, 'n inkubasie tydperk van 15 dae en temperatuur van 30°C. Drie melksuurbakteriee (226 - Lactobacillus plantarum, 140 - Lactococcus diacetylactis en 407 - Pediococcus cerevisiae) is gekies aangesien hulle die beste resultate gelewer het en veilig vir gebruik in voedselprodukte is. Daar is gevind dat die drie melksuurkulture saam in kombinasie die beste fermentasie resultate opgelewer het. Deur gebruik te maak van die bogenoemde fermentasie kondisies, kon 'n aanvaarbare produk, in terme van kleur en geur, gelewer word. Verdere studies moet gedoen word om die veiligheid asook die geur, chemies asook sensories, te optimiseer.

Thesis (MScFoodSc)--Stellenbosch University, 2001. / ENGLISH ABSTRACT: The preservation of various fresh fish products is achieved by either smoking, salting, canning, freezing or fermenting a highly perishable raw product. Since many of these facilities are not readily available, the use of fermentation as a means of preserving the product has been extensively practiced. However, the fermentation of fish is a time consuming practise and only by accelerating the process would it be possible to ensure the production of a more cost effective and readily available safe end-product. The quality of the fermented fish product is partially determined by the fermentation conditions and the metabolic activity of the microbes present. The rapid identification of the microbes present during the fermentation would enable the selection of possible starters to ensure an accelerated production of high quality fermented fish products. This study was thus undertaken to develop identification fingerprints for bacteria isolated from fermented fish products. A 1300 bp fragment of the 16S rRNA genes of each of the bacteria previously isolated was successfully amplified using the PCR technique. The isolates included strains of the genera Bacillus, Staphylococcus, Sphingomonas, Kocuria, Brevibacillus, Cryseomonas, Vibrio, Stenotrophomonas and Agrobacterium. The data obtained can, therefore, be used in the identification of these microbes isolated from other similar fermented fish products. The fingerprints could also be used to assist in determining the dominant microbial populations responsible for the characteristic qualitative changes occurring in the fish product during fermentation. The microbial composition of a fermenting fish product partially determines the quality of the end-product, therefore, the use of selected bacterial starters could result in the accelerated production of a microbial safe fermented fish product. A further objective of this study was to accelerate the production of a fermented fish product by inoculating macerated trout with either selected lactic acid bacteria (LAB) or with selected bacteria with high proteolytic activity over a 30 day fermentation period. The LAB included a combination of Lactobacillus plantarum, Lactococcus diacetylactis and Pediococcus cerevisiae strains, whereas the bacteria with high proteolytic activity included strains of Kocuria varians, Bacillus subtilis, two strains of B. amyloliquefaciens and a combination of these bacterial species. The quality of the fermented product was determined using changes in product pH, titratable acidity (%TA) and free amino nitrogen (FAN) formation as efficiency parameters. The data obtained during the fermentation of the macerated trout showed that the selected starters did not have a significant effect on the pH decrease in the product over a 30 day fermentation period. The LAB strains did not have a significant effect on the %TA of the fermenting fish product, yet the presence of these bacteria appeared to limit the FAN production in the product. The bacteria with high proteolytic activity resulted in slightly enhanced %TA values and a higher FAN content in the fermented product. It was also determined that the LAB and Kocuria varians, in contrast to the Bacillus spp. inoculums, did not survive the fermentation conditions well, possibly due to the low pH environment. The presence of the starter bacteria in the fermenting fish mixture at the end of the fermentation was also successfully determined with the use of the PCR-RFLP technique. The fermented fish product, obtained at the end of the fermentation period, had a good aroma and compared favourably to similar commercially available fermented fish products. The use of different microbial starters could in future enable the production of a diverse range of high quality products, which could be produced and marketed locally. / AFRIKAANSE OPSOMMING: Die preservering van ‘n verskeidenheid vars vis produkte word bereik deur die hoogs bederfbare produk te rook, te sout, te blik, te vries of te fermenteer. Aangesien baie van hierdie fasiliteite nie geredelik beskikbaar is nie, is die gebruik van fermentasie as ‘n preserverings metode al ekstensief beoefen. Die fermentasie van vis is egter 'n tydsame proses en slegs deur die versnelling van die proses sal dit moontlik wees om die produksie van ‘n meer koste effektiewe en geredelike beskikbare veilige eindproduk te verseker. Die kwaliteit van die gefermenteerde vis produk word gedeeltelik bepaal deur die fermentasie kondisies en die metaboliese aktiwiteit van die mikrobes teenwoordig. Die vinnige identifikasie van die mikrobes teenwoordig gedurende die fermentasie sal die seleksie van moontlike suursels om die versnelde produksie van hoë kwaliteit gefermenteerde vis produkte moontlik maak. Hierdie studie is dus onderneem om identifikasie vingerafdrukke vir bakteriee wat gei'soleer is van gefermenteerde vis produkte moontlik te maak. ‘n 1300 bp fragment van die 16S rRNA gene van elkeen van die bakteriee wat voorheen gei'soleer is, is suksesvol geamplifiseer deur die PCR tegniek. Die isolate sluit in stamme van die genera Bacillus, Staphylococcus, Sphingomonas, Kocuria, Brevibacillus, Cryseomonas, Vibrio, Stenotrophomonas en Agrobacterium. Die data kan dus gebruik word in die identifikasie van hierdie mikrobes as dit gei'soleer word van ander gefermenteerde vis produkte. Die vingerafdrukke kan ook gebruik word om die dominante mikrobiese populasies wat verantwoordelik is vir die kenmerklike kwalitatiewe veranderinge wat plaasvind in die vis produk gedurende die fermentasie, te identifiseer. Die mikrobiese samestelling van ‘n fermenterende vis produk bepaal gedeeltelik die kwaliteit van die eindproduk, daarom kan die gebruik van geselekteerde bakteriese suursels die versnelde produksie van ‘n mikrobies veilige gefermenteerde vis produk teweeg bring. ‘n Verdere doel van hierdie studie was om die produksie van ‘n gefermenteerde vis produk te versnel deur fyngemaakte forel met of geselekteerde melksuurbakteriee of met geselekteerde bakteriee met hoë proteolitiese aktiwiteit te inokuleer oor ‘n 30 dag fermentasie periode. Die melksuurbakteriee het ingesluit ‘n kombinasie van Lactobacillus plantarum, Lactococcus diacetylactis en Pediococcus cerevisiae, terwyl die bakterieë met hoë proteolitiese aktiwiteit stamme van Kocuria varians, Bacillus subtilis, twee stamme van Bacillus amyloliquefaciens en ‘n kombinasie van hierdie bakteriese stamme ingesluit het. Die kwaliteit van die gefermenteerde produk is bepaal deur die veranderinge in die pH, titreerbare suur (%TS) en vrye amino stikstof (VAS) vorming van die produk as effektiwiteits parameters te gebruik. Die data wat verkry is gedurende die fermentasie van die fyngemaakte forel het gedui daarop dat die geselekteerde suursels nie ‘n merkwaardige effek op die afname in pH in die produk oor ‘n 30 dag fermentasie periode het nie. Die melksuurbakteriee het nie ‘n merkwaardige effek op die %TS van die gefermenteerde vis produk gehad nie, terwyl dit geblyk het dat die teenwoordigheid van hierdie bakterieë die produksie van VAS in die produk belemmer het. Die bakteriee met hoe proteolitiese aktiwiteit het ‘n effense verhoogde %TS en ‘n hoër VAS inhoud in die gefermenteerde produk veroorsaak. Dit is ook bepaal dat die melksuurbakteriee en Kocuria varians, in teenstelling met die Bacillus spp. inokulums, nie die fermentasie kondisies goed oorleef het nie, moontlik as gevolg van die lae pH omgewing. Die teenwoordigheid van die suursel bakteriee in die fermenterende vis mengsel aan die einde van die fermentasie is ook suksesvol bepaal met die PKR-RFLP tegniek. Die gefermenteerde vis produk, verkry aan die einde van die fermentasie periode, het ‘n goeie aroma gehad en het goed vergelyk met soortgelyke kommersieel beskikbare gefermenteerde vis produkte. Die gebruik van verskillende mikrobiese suursels kan in die toekoms die produksie van ‘n diverse reeks hoë kwaliteit produkte wat plaaslik geproduseer en bemark kan word moontlik maak.

Background. Traditional plant use was studied in Lao PDR. Research focused on medicinal plant use by the Brou, Saek and Kry ethnic groups, traditional plant repellents against parasitic arthropods and leeches, and the phylogeny and biogeography of the medicinally-important snake gourd genus (Trichosanthes, Cucurbitaceae).  Methods. The ethnobiology research used a combination of structured interviews, village surveys, botanical collecting, hydro-distillation, GC-MS analysis, literature studies, and laboratory experiments. The plant systematics research used a combination of morphological studies, molecular biology laboratory work, and phylogenetic, dating and biogeographical analysis.  Results. Informants reported the use of close to 100 species to repel arthropods and leeches, many of which have constituents with documented efficacy.  Brou, Saek and Kry informants use over 75 plant species for women’s healthcare, mainly during the postpartum period for steam sauna, steam bath, hotbed, mother roasting, medicinal decoctions and infusions, and postpartum diet.  A molecular phylogeny of Trichosanthes and Gymnopetalum using a broad sampling of ~60% of their species and 4756 nucleotides of nuclear and plastid DNA shows that Gymnopetalum is nested within Trichosanthes. Fossil-calibrated Bayesian molecular dating of the Trichosanthes phylogeny reveals an early Oligocene origin of the genus, and many of the extant sections originating and diversifying during the Miocene. Biogeographical analysis shows a likely East or South Asian origin of Trichosanthes, with lineages diversifying and spreading throughout Australasia from the early Pliocene to the Pleistocene.  Discussion. Traditional plant use in Lao PDR is common and widespread. The presence among the repellent species of economical alternatives to costly synthetic repellents is tenable, and the subject of ongoing studies.  Postpartum traditions and medicinal plant use are essential parts of childbirth and postpartum recovery in these ethnic groups, and many other groups in Lao PDR. Efforts to improve maternal healthcare and reduce maternal and infant mortality need to integrate these traditions with modern notions of healthcare to achieve wider adoption. Documenting all possible uses of commonly used medicinal plant species shows that similarity in use between these ethnic groups is relatively low considering that they share, and have shared for many generations, the same environment and resources. A lack of effective cures leads to a process of continuous innovation, where effective cures are shared between cultures, but remedies of only cultural importance, or those under evaluation are culture-specific.  The Trichosanthes phylogeny implies the merging of Gymnopetalum into Trichosanthes, and this is done using available names or new combinations. A synopsis of Trichosanthes, the new combinations, and a revision of the species in Australia, are made and presented.  Conclusions. Traditional plant use is widespread in Lao PDR, and of significance to many people as a source of primary healthcare and inexpensive repellents. The important medicinal plant genus Trichosanthes includes Gymnopetalum, and has a complex biogeographic history with multiple colonization events of Australasia.